U.S. patent number 8,067,549 [Application Number 12/258,337] was granted by the patent office on 2011-11-29 for humanized antibodies and compositions for binding sphingosine-1-phosphate.
This patent grant is currently assigned to Lpath, Inc.. Invention is credited to William A. Garland, Genevieve Hansen, Steven Tarran Jones, Roger A. Sabbadini, David Gareth Williams.
United States Patent |
8,067,549 |
Sabbadini , et al. |
November 29, 2011 |
Humanized antibodies and compositions for binding
sphingosine-1-phosphate
Abstract
The present invention relates to anti-S1P agents, particularly
humanized monoclonal antibodies (and antigen binding fragments
thereof) specifically reactive with S1P, compositions containing
such antibodies (or fragments), and the use of such antibodies (or
fragments), for example, to treat diseases and conditions
associated with aberrant levels of S1P.
Inventors: |
Sabbadini; Roger A. (Lakeside,
CA), Garland; William A. (San Clemente, CA), Hansen;
Genevieve (San Diego, CA), Jones; Steven Tarran
(Radlett, GB), Williams; David Gareth (Epsom,
GB) |
Assignee: |
Lpath, Inc. (San Diego,
CA)
|
Family
ID: |
39492938 |
Appl.
No.: |
12/258,337 |
Filed: |
October 24, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090148443 A1 |
Jun 11, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11924890 |
Oct 26, 2007 |
7829674 |
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60854971 |
Oct 27, 2006 |
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Current U.S.
Class: |
530/387.3;
424/133.1; 424/130.1; 530/387.1; 424/134.1; 530/388.1 |
Current CPC
Class: |
A61P
35/04 (20180101); A61P 9/10 (20180101); A61P
11/00 (20180101); A61P 29/00 (20180101); A61P
35/02 (20180101); A61P 25/00 (20180101); A61P
37/06 (20180101); A61P 43/00 (20180101); A61P
9/00 (20180101); A61P 17/00 (20180101); C07K
16/44 (20130101); A61P 11/06 (20180101); A61P
27/02 (20180101); A61P 17/02 (20180101); A61P
27/06 (20180101); A61P 9/04 (20180101); A61P
35/00 (20180101); A61P 37/02 (20180101); C07K
2317/56 (20130101); C07K 2317/565 (20130101); A61K
2039/505 (20130101); C07K 2317/24 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); C12P 21/08 (20060101); A61K
39/395 (20060101); A61K 39/00 (20060101) |
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|
Primary Examiner: Gussow; Anne M.
Attorney, Agent or Firm: Acuity Law Group, P.C. Chambers;
Daniel M.
Parent Case Text
RELATED APPLICATION
This application claims the benefit of and priority to U.S.
provisional application Ser. No. 60/854,971, filed on Oct. 27,
2006, and U.S. non-provisional patent application Ser. No.
11/924,890, filed 26 Oct. 2007, the contents of each of which are
herein incorporated by reference in their entirety for any and all
purposes.
Claims
What is claimed is:
1. An isolated humanized antibody, or an antigen binding fragment
thereof, that binds sphingosine-1-phosphate (S1P) and comprising at
least one heavy chain variable domain and at least one light chain
variable domain, wherein: A. each heavy chain variable domain
comprises: (i) a first sequence of amino acid residues of sequence
DHTIH (SEQ ID NO: 13); (ii) a second sequence of amino acid
residues AISPRHDITKYNEMFRG (SEQ ID NO: 31); and (iii) a third
sequence of amino acid residues of sequence GGFYGSTIWFDF (SEQ ID
NO: 15); and B. each light chain variable domain comprises: (i) a
first sequence of amino acid residues of sequence ITTTDIDDDMN (SEQ
ID NO: 10); (ii) a second sequence of amino acid residues of
sequence EGNILRP (SEQ ID NO: 11); and (iii) a third sequence of
amino acid residues of sequence LQSDNLPFT SEQ ID NO: 12).
2. An isolated humanized antibody, or an antigen binding fragment
thereof, according to claim 1, wherein: A. each heavy chain
variable domain comprises a sequence of amino acid residues having
an amino acid sequence TABLE-US-00015 (SEQ ID NO: 32, residues
20-140, inclusive) EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWM
GAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCA
RGGFYGSTIWFDFWGQGTMVTVSS; and
B. each light chain variable domain comprises a sequence of amino
acid residues having an amino acid sequence TABLE-US-00016 (SEQ ID
NO: 33, residues 21-127, inclusive)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPG
KAPKLLISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDF
ATYYCLQSDNLPFTFGQGTKLEIK.
3. An isolated humanized antibody according to claim 1, wherein: A.
at least one heavy chain comprises a sequence of amino acid
residues having an amino acid sequence: TABLE-US-00017 (SEQ ID NO:
38, residues 20-455, inclusive)
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE
WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMY
FCARGGFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK; and
B. at least one light chain comprises a sequence of amino acid
residues having an amino acid sequence: TABLE-US-00018 (SEQ ID NO:
37, residues 21-234, inclusive)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKL
LISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPF
TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC.
4. An isolated humanized antibody according to claim 1, wherein: A.
each heavy chain comprises a sequence of amino acid residues having
an amino acid sequence: TABLE-US-00019 (SEQ ID NO: 38, residues
20-455, inclusive) EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE
WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMY
FCARGGFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPG; and
B. each light chain comprises a sequence of amino acid residues
having an amino acid sequence: TABLE-US-00020 (SEQ ID NO: 37,
residues 21-234, inclusive)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKL
LISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPF
TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC.
5. An isolated humanized antibody, or an antigen binding fragment
thereof, according to claim 1, wherein: A. each heavy chain
variable domain comprises the same heavy chain variable domain
amino acid sequence as the heavy chain variable domain of the
antibody that binds S1P encoded by the heavy chain structural gene
in vector pATH1009 in ATCC Accession No. PTA-8421; and B. each
light chain variable domain comprises the same light chain variable
domain amino acid sequence as the light chain variable domain of
the antibody that binds S1P encoded by the light chain structural
gene in vector pATH1009 in ATCC Accession No. PTA-8421.
6. An isolated humanized antibody, or an antigen binding fragment
thereof, according to claim 1 wherein at least one amino acid
residue of the antibody or antigen binding fragment is
glycosylated.
7. An isolated humanized antibody according to claim 1.
8. An isolated humanized antibody according to claim 1 that
comprises two heavy chains and two light chains.
9. An isolated humanized antibody according to claim 1, obtainable
from CHO cell line LH1 275, as deposited under accession number
ATCC PTA-8422.
10. An isolated humanized antibody according to claim 1 as produced
in mammalian cells transfected with the plasmid pATH1009 obtainable
from E. coli StB12, as deposited under accession number ATCC
PTA-8421.
11. An isolated humanized antibody, or an antigen binding fragment
thereof, according to claim 1, wherein: A. each heavy chain
variable domain comprises the same heavy chain variable domain
amino acid sequence as the heavy chain variable domain of the
antibody expressed by CHO cell line LH1 275, as deposited under
accession number ATCC PTA-8422; and B. each light chain variable
domain comprises the same light chain variable domain amino acid
sequence as the light chain variable domain of the antibody
expressed by CHO cell line LH1 275, as deposited under accession
number ATCC PTA-8422.
12. A pharmaceutical composition comprising an isolated humanized
antibody, or an antigen binding fragment thereof, according to
claim 1 and a pharmaceutically acceptable carrier.
Description
SEQUENCE LISTING
This application has been filed with, and includes, the sequence
listing concurrently submitted herewith, which sequence listing has
been prepared and filed in accordance with applicable regulations
and procedures. This sequence listing is hereby incorporated by
reference for any and all purposes.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to agents that bind
sphingosine-1-phosphate (S1P), particularly to humanized monoclonal
antibodies, antibody fragments, and antibody derivatives
specifically reactive to S1P under physiological conditions. Such
agents can be used in the treatment and/or prevention of various
diseases or disorders through the delivery of pharmaceutical
compositions that contain such agents.
The following description includes information that may be useful
in understanding the present invention. It is not an admission that
any of the information provided herein, or any publication
specifically or implicitly referenced herein, is prior art, or even
particularly relevant, to the presently claimed invention.
2. Background
Bioactive Signaling Lipids
Lipids and their derivatives are now recognized as important
targets for medical research, not as just simple structural
elements in cell membranes or as a source of energy for
.beta.-oxidation, glycolysis or other metabolic processes. In
particular, certain bioactive lipids function as signaling
mediators important in animal and human disease. Although most of
the lipids of the plasma membrane play an exclusively structural
role, a small proportion of them are involved in relaying
extracellular stimuli into cells. "Lipid signaling" refers to any
of a number of cellular signal transduction pathways that use cell
membrane lipids as second messengers, as well as referring to
direct interaction of a lipid signaling molecule with its own
specific receptor. Lipid signaling pathways are activated by a
variety of extracellular stimuli, ranging from growth factors to
inflammatory cytokines, and regulate cell fate decisions such as
apoptosis, differentiation and proliferation. Research into
bioactive lipid signaling is an area of intense scientific
investigation as more and more bioactive lipids are identified and
their actions characterized.
Examples of bioactive lipids include the eicosanoids (including the
cannabinoids, leukotrienes, prostaglandins, lipoxins,
epoxyeicosatrienoic acids, and isoeicosanoids), non-eicosanoid
cannabinoid mediators, phospholipids and their derivatives such as
phosphatidic acid (PA) and phosphatidylglycerol (PG), platelet
activating factor (PAF) and cardiolipins as well as
lysophospholipids such as lysophosphatidyl choline (LPC) and
various lysophosphatidic acids (LPA). Bioactive signaling lipid
mediators also include the sphingolipids such as sphingomyelin,
ceramide, ceramide-1-phosphate, sphingosine, sphingosylphosphoryl
choline, sphinganine, sphinganine-1-phosphate (Dihydro-S1P) and
sphingosine-1-phosphate. Sphingolipids and their derivatives
represent a group of extracellular and intracellular signaling
molecules with pleiotropic effects on important cellular processes.
Other examples of bioactive signaling lipids include
phosphatidylserine (PS), phosphatidylinositol (PI),
phosphatidylethanolamine (PEA), diacylglyceride (DG), sulfatides,
gangliosides, and cerebrosides.
Sphingolipids are a unique class of lipids that were named, due to
their initially mysterious nature, after the Sphinx. Sphingolipids
were initially characterized as primary structural components of
cell membranes, but recent studies indicate that sphingolipids also
serve as cellular signaling and regulatory molecules (Hannun, et
al., Adv. Lipid Res. 25:27-41, 1993; Speigel, et al., FASEB J.
10:1388-1397, 1996; Igarashi, J. Biochem 122:1080-1087, 1997; Hla,
T. (2004). Semin Cell Dev Biol, 15, 513-2; Gardell, S. E., Dubin,
A. E. & Chun, J. (2006). Trends Mol Med, 12, 65-75).
Sphingolipids are primary structural components of cell membranes
that also serve as cellular signaling and regulatory molecules
(Hannun and Bell, Adv. Lipid Res. 25: 27-41, 1993; Igarashi, J.
Biochem 122: 1080-1087, 1997). The sphingolipid signaling
mediators, ceramide (CER), sphingosine (SPH) and
sphingosine-1-phosphate (S1P), have been most widely studied and
have recently been appreciated for their roles in the
cardiovascular system, angiogenesis and tumor biology (Claus, et
al., Curr Drug Targets 1: 185-205, 2000; Levade, et al., Circ. Res.
89: 957-968, 2001; Wang, et al., J. Biol. Chem. 274: 35343-50,
1999; Wascholowski and Giannis, Drug News Perspect. 14: 581-90,
2001; Spiegel, S. & Milstien, S. (2003).
Sphingosine-1-phosphate: an enigmatic signaling lipid. Nat Rev Mol
Cell Biol, 4, 397-407).
For a review of sphingolipid metabolism, see Liu, et al., Crit Rev.
Clin. Lab. Sci. 36:511-573, 1999. For reviews of the sphingomyelin
signaling pathway, see Hannun, et al., Adv. Lipid Res. 25:27-41,
1993; Liu, et al., Crit. Rev. Clin. Lab. Sci. 36:511-573, 1999;
Igarashi, J. Biochem. 122:1080-1087, 1997; Oral, et al., J. Biol.
Chem. 272:4836-4842, 1997; and Spiegel et al., Biochemistry
(Moscow) 63:69-83, 1998.
S1P is a mediator of cell proliferation and protects from apoptosis
through the activation of survival pathways (Maceyka, et al.
(2002), BBA, vol. 1585): 192-201, and Spiegel, et al. (2003),
Nature Reviews Molecular Cell Biology, vol. 4: 397-407). It has
been proposed that the balance between CER/SPH levels and S1P
provides a rheostat mechanism that decides whether a cell is
directed into the death pathway or is protected from apoptosis. The
key regulatory enzyme of the rheostat mechanism is sphingosine
kinase (SPHK) whose role is to convert the death-promoting
bioactive signaling lipids (CER/SPH) into the growth-promoting S1P.
S1P has two fates: S1P can be degraded by S1P lyase, an enzyme that
cleaves S1P to phosphoethanolamine and hexadecanal, or, less
common, hydrolyzed by S1P phosphatase to SPH.
The pleiotropic biological activities of S1P are mediated via a
family of G protein-coupled receptors (GPCRs) originally known as
Endothelial Differentiation Genes (EDG). Five GPCRs have been
identified as high-affinity S1P receptors (S1PRs): S1P.sub.1/EDG-1,
S1P.sub.2/EDG-5, S1P.sub.3/EDG-3, S1P.sub.4/EDG-6, and
S1P.sub.5/EDG-8 only identified as late as 1998 (Lee, et al.,
1998). Many responses evoked by S1P are coupled to different
heterotrimeric G proteins (G.sub.q-, G.sub.i, G.sub.12-13) and the
small GTPases of the Rho family (Gardell, et al., 2006).
In the adult, S1P is released from platelets (Murata et al., 2000)
and mast cells to create a local pulse of free S1P (sufficient
enough to exceed the K.sub.d of the S1PRs) for promoting wound
healing and participating in the inflammatory response. Under
normal conditions, the total S1P in the plasma is quite high
(300-500 nM); however, it has been hypothesized that most of the
S1P may be `buffered` by serum proteins, particularly lipoproteins
(e.g., HDL>LDL>VLDL) and albumin, so that the bio-available
S1P (or the free fraction of S1P) is not sufficient to appreciably
activate S1PRs (Murata et al., 2000). If this were not the case,
inappropriate angiogenesis and inflammation would result.
Intracellular actions of S1P have also been suggested (see, e.g.,
Spiegel S, Kolesnick R (2002), Leukemia, vol. 16: 1596-602;
Suomalainen, et al (2005), Am J Pathol, vol. 166: 773-81).
Widespread expression of the cell surface S1P receptors allows S1P
to influence a diverse spectrum of cellular responses, including
proliferation, adhesion, contraction, motility, morphogenesis,
differentiation, and survival. This spectrum of response appears to
depend upon the overlapping or distinct expression patterns of the
S1P receptors within the cell and tissue systems. In addition,
crosstalk between S1P and growth factor signaling pathways,
including platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), and basic fibroblastic growth
factor (bFGF), have recently been demonstrated (see, e.g.,
Baudhuin, et al. (2004), FASEB J, vol. 18: 341-3). The regulation
of various cellular processes involving S1P has particular impact
on neuronal signaling, vascular tone, wound healing, immune cell
trafficking, reproduction, and cardiovascular function, among
others. Alterations of endogenous levels of S1P within these
systems can have detrimental effects, eliciting several
pathophysiological conditions, including cancer, inflammation,
angiogenesis, heart disease, asthma, and autoimmune diseases.
A recent novel approach to the treatment of various diseases and
disorders, including cardiovascular diseases, cerebrovascular
diseases, and various cancers, involves reducing levels of
biologically available S1P, either alone or in combination with
other treatments. While sphingolipid-based treatment strategies
that target key enzymes of the sphingolipid metabolic pathway, such
as SPHK, have been proposed, interference with the lipid mediator
S1P itself has not until recently been emphasized, largely because
of difficulties in directly mitigating this lipid target, in
particular because of the difficulty first in raising and then in
detecting antibodies against the S1P target.
Recently, the generation of antibodies specific for S1P has been
described. See, e.g., commonly owned, U.S. patent application
Serial No. 20070148168; WO2007/053447. Such antibodies, which can,
for example, selectively adsorb S1P from serum, act as molecular
sponges to neutralize extracellular S1P. See also commonly owned
U.S. Pat. Nos. 6,881,546 and 6,858,383 and U.S. patent application
Ser. No. 10/029,372. SPHINGOMAB.TM., the murine monoclonal antibody
(mAb) developed by Lpath, Inc. and described in certain patents or
patent applications listed above, has been shown to be effective in
models of human disease. In some situations, a humanized antibody
may be preferable to a murine antibody, particularly for
therapeutic uses in humans, where human-anti-mouse antibody (HAMA)
response may occur. Such a response may reduce the effectiveness of
the antibody by neutralizing the binding activity and/or by rapidly
clearing the antibody from circulation in the body. The HAMA
response can also cause toxicities with subsequent administrations
of mouse antibodies.
A humanized anti-S1P antibody has now been developed and is
described herein. This antibody is expected to have all the
advantages of the murine mAb in terms of efficacy in binding S1P,
neutralizing S1P and modulating disease states related to S1P, but
with none of the potential disadvantages of the murine mAb when
used in a human context. As described in the examples hereinbelow,
this humanized antibody (referred to as LT1009 or sonepcizumab) has
in fact shown activity greater than that of the parent (murine)
antibody in animal models of disease.
3. Definitions
Before describing the instant invention in detail, several terms
used in the context of the present invention will be defined. In
addition to these terms, others are defined elsewhere in the
specification, as necessary. Unless otherwise expressly defined
herein, terms of art used in this specification will have their
art-recognized meanings. In the event of conflict, the present
specification, including definitions, will control.
An "immune-derived moiety" includes any antibody (Ab) or
immunoglobulin (Ig), and refers to any form of a peptide,
polypeptide derived from, modeled after or encoded by, an
immunoglobulin gene, or a fragment of such peptide or polypeptide
that is capable of binding an antigen or epitope (see, e.g.,
Immunobiology, 5th Edition, Janeway, Travers, Walport, Shlomchiked.
(editors), Garland Publishing (2001)). In the present invention,
the antigen is a bioactive lipid molecule.
An "anti-S1P antibody" or an "immune-derived moiety reactive
against S1P" refers to any antibody or antibody-derived molecule
that binds S1P. As will be understood from these definitions,
antibodies or immune-derived moieties may be polyclonal or
monoclonal and may be generated through a variety of means, and/or
may be isolated from an animal, including a human subject.
A "bioactive lipid" refers to a lipid signaling molecule. In
general, a bioactive lipid does not reside in a biological membrane
when it exerts its signaling effects, which is to say that while
such a lipid species may exist at some point in a biological
membrane (for example, a cell membrane, a membrane of a cell
organelle, etc.), when associated with a biological membrane it is
not a "bioactive lipid" but is instead a "structural lipid"
molecule. Bioactive lipids are distinguished from structural lipids
(e.g., membrane-bound phospholipids) in that they mediate
extracellular and/or intracellular signaling and thus are involved
in controlling the function of many types of cells by modulating
differentiation, migration, proliferation, secretion, survival, and
other processes. In vivo, bioactive lipids can be found in
extracellular fluids, where they can be complexed with other
molecules, for example serum proteins such as albumin and
lipoproteins, or in "free" form, i.e., not complexed with another
molecule species. As extracellular mediators, some bioactive lipids
alter cell signaling by activating membrane-bound ion channels or
G-protein coupled receptors that, in turn, activate complex
signaling systems that result in changes in cell function or
survival. As intracellular mediators, bioactive lipids can exert
their actions by directly interacting with intracellular components
such as enzymes and ion channels. Representative examples of
bioactive lipids include LPA and S1P.
The term "therapeutic agent" means an agent to mitigate
angiogenesis and/or neovascularization, e.g., CNV and BV
maturation, edema, vascular permeability and fibrosis, fibrogenesis
and scarring associated with, or part of the underlying pathology
of, ocular diseases and conditions.
The term "combination therapy" refers to a therapeutic regimen that
involves the provision of at least two distinct therapies to
achieve an indicated therapeutic effect. For example, a combination
therapy may involve the administration of two or more chemically
distinct active ingredients, for example, an anti-LPA antibody and
an anti-S1P antibody. Alternatively, a combination therapy may
involve the administration of an immune-derived moiety reactive
against a bioactive lipid and the administration of one or more
other chemotherapeutic agents. Combination therapy may,
alternatively, involve administration of an anti-lipid antibody
together with the delivery of another treatment, such as radiation
therapy and/or surgery. Further, a combination therapy may involve
administration of an anti-lipid antibody together with one or more
other biological agents (e.g., anti-VEGF, TGF.beta., PDGF, or bFGF
agent), chemotherapeutic agents and another treatment such as
radiation and/or surgery. In the context of combination therapy
using two or more chemically distinct active ingredients, it is
understood that the active ingredients may be administered as part
of the same composition or as different compositions. When
administered as separate compositions, the compositions comprising
the different active ingredients may be administered at the same or
different times, by the same or different routes, using the same of
different dosing regimens, all as the particular context requires
and as determined by the attending physician. Similarly, when one
or more anti-lipid antibody species, for example, an anti-LPA
antibody, alone or in conjunction with one or more chemotherapeutic
agents are combined with, for example, radiation and/or surgery,
the drug(s) may be delivered before or after surgery or radiation
treatment.
An "anti-S1P agent" refers to any agent that is specifically
reactive to S1P, and includes antibodies or antibody-derived
molecules or non-antibody-derived moieties that bind S1P and its
variants.
A "hapten" refers to a molecule adapted for conjugation to a
hapten, thereby rendering the hapten immunogenic. A representative,
non-limiting class of hapten molecules is proteins, examples of
which include albumin, keyhole limpet hemocyanin, hemaglutanin,
tetanus, and diphtheria toxoid. Other classes and examples of
hapten molecules suitable for use in accordance with the invention
are known in the art. These, as well as later discovered or
invented naturally occurring or synthetic haptens, can be adapted
for application in accordance with the invention.
The term "chemotherapeutic agent" means anti-cancer and other
anti-hyperproliferative agents. Put simply, a "chemotherapeutic
agent" refers to a chemical intended to destroy cells and tissues.
Such agents include, but are not limited to: (1) DNA damaging
agents and agents that inhibit DNA synthesis: anthracyclines
(doxorubicin, donorubicin, epirubicin), alkylating agents
(bendamustine, busulfan, carboplatin, carmustine, cisplatin,
chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine,
ifosphamide, lomustine, mechlorethamine, melphalan, mitotane,
mytomycin, pipobroman, procarbazine, streptozocin, thiotepa, and
triethylenemelamine), platinum derivatives (cisplatin, carboplatin,
cis diamminedichloroplatinum), telomerase and topoisomerase
inhibitors (Camptosar), (2) tubulin-depolymerizing agents: taxoids
(Paclitaxel, docetaxel, BAY 59-8862), (3) anti-metabolites such as
capecitabine, chlorodeoxyadenosine, cytarabine (and its activated
form, ara-CMP), cytosine arabinoside, dacabazine, floxuridine,
fludarabine, 5-fluorouracil, 5-DFUR, gemcitibine, hydroxyurea,
6-mercaptopurine, methotrexate, pentostatin, trimetrexate, and
6-thioguanine (4) anti-angiogenics (Avastin, thalidomide,
sunitinib, lenalidomide), vascular disrupting agents
(flavonoids/flavones, DMXAA, combretastatin derivatives such as
CA4DP, ZD6126, AVE8062A, etc.), (5) biologics such as antibodies or
antibody fragments (Herceptin, Avastin, Panorex, Rituxan, Zevalin,
Mylotarg, Campath, Bexar, Erbitux, Lucentis), and (6) endocrine
therapy: aromatase inhibitors (4-hydroandrostendione, exemestane,
aminoglutehimide, anastrozole, letozole), anti-estrogens
(Tamoxifen, Toremifine, Raoxifene, Faslodex), steroids such as
dexamethasone, (7) immuno-modulators: cytokines such as IFN-beta
and IL2), inhibitors to integrins, other adhesion proteins and
matrix metalloproteinases), (8) histone deacetylase inhibitors, (9)
inhibitors of signal transduction such as inhibitors of tyrosine
kinases like imatinib (Gleevec), (10) inhibitors of heat shock
proteins, (11) retinoids such as all trans retinoic acid, (12)
inhibitors of growth factor receptors or the growth factors
themselves, (13) anti-mitotic compounds such as navelbine,
Paclitaxel, taxotere, vinblastine, vincristine, vindesine, and
vinorelbine, (14) anti-inflammatories such as COX inhibitors and
(15) cell cycle regulators, e.g., check point regulators and
telomerase inhibitors.
The term "sphingolipid" as used herein refers to the class of
compounds in the art known as sphingolipids, including, but not
limited to the following compounds (see lipidmaps.org as the site
containing the links indicated by the bracketed alphanumeric
strings below, which links contain chemical formulas, structural
information, etc. for the corresponding compounds):
Sphingoid bases [SP01] Sphing-4-enines (Sphingosines) [SP0101]
Sphinganines [SP0102] 4-Hydroxysphinganines (Phytosphingosines)
[SP0103] Sphingoid base homologs and variants [SP0104] Sphingoid
base 1-phosphates [SP0105] Lysosphingomyelins and
lysoglycosphingolipids [SP0106] N-methylated sphingoid bases
[SP0107] Sphingoid base analogs [SP0108]
Ceramides [SP02] N-acylsphingosines (ceramides) [SP0201]
N-acylsphinganines (dihydroceramides) [SP0202]
N-acyl-4-hydroxysphinganines (phytoceramides) [SP0203]
Acylceramides [SP0204] Ceramide 1-phosphates [SP0205]
Phosphosphingolipids [SP03] Ceramide phosphocholines
(sphingomyelins) [SP0301] Ceramide phosphoethanolamines [SP0302]
Ceramide phosphoinositols [SP0303]
Phosphonosphingolipids [SP04]
Neutral glycosphingolipids [SP05] Simple Glc series (GlcCer,
LacCer, etc) [SP0501] GalNAcb1-3Gala1-4Galb1-4Glc- (Globo series)
[SP0502] GalNAcb1-4Galb1-4Glc-(Ganglio series) [SP0503]
Galb1-3GlcNAcb1-3Galb1-4Glc-(Lacto series) [SP0504]
Galb1-4GlcNAcb1-3Galb1-4Glc-(Neolacto series) [SP0505]
GalNAcb1-3Gala1-3Galb1-4Glc-(Isoglobo series) [SP0506]
GlcNAcb1-2Mana1-3Manb1-4Glc-(Mollu series) [SP0507]
GalNAcb1-4GlcNAcb1-3Manb1-4Glc-(Arthro series) [SP0508] Gal-(Gala
series) [SP0509] Other [SP0510]
Acidic glycosphingolipids [SP06] Gangliosides [SP0601]
Sulfoglycosphingolipids (sulfatides) [SP0602]
Glucuronosphingolipids [SP0603] Phosphoglycosphingolipids [SP0604]
Other [SP0600]
Basic glycosphingolipids [SP07]
Amphoteric glycosphingolipids [SP08]
Arsenosphingolipids [SP09]
The present invention provides anti-sphingolipid S1P agents that
are useful for treating or preventing hyperproliferative disorders
such as cancer and cardiovascular or cerebrovascular diseases and
disorders and various ocular disorders, as described in greater
detail below. In particular the invention is drawn to S1P and its
variants including but are not limited to sphingosine-1-phosphate
[sphingene-1-phosphate; D-erythro-sphingosine-1-phosphate;
sphing-4-enine-1-phosphate;
(E,2S,3R)-2-amino-3-hydroxy-octadec-4-enoxy]phosphonic acid (AS
26993-30-6), DHS1P is defined as dihydrosphingosine-1-phosphate
[sphinganine-1-phosphate;
[(2S,3R)-2-amino-3-hydroxy-octadecoxy]phosphonic acid;
D-Erythro-dihydro-D-sphingosine-1-phosphate (CAS 19794-97-9]; SPC
is sphingosylphosphoryl choline, lysosphingomyelin,
sphingosylphosphocholine, sphingosine phosphorylcholine,
ethanaminium;
2-((((2-amino-3-hydroxy-4-octadecenyl)oxy)hydroxyphosphinyl)oxy)-N,N,N-tr-
imethyl-, chloride, (R-(R*,S*-(E))), 2-[[(E,2R,3
S)-2-amino-3-hydroxy-octadec-4-enoxy]-hydroxy-phosphoryl]oxyethyl-trimeth-
yl-azanium chloride (CAS 10216-23-6).
The term "epitope" or "antigenic determinant" when used herein,
unless indicated otherwise, refers to the region of S1P to which an
anti-S1P agent is reactive to.
The term "hyperproliferative disorder" refers to diseases and
disorders associated with, the uncontrolled proliferation cells,
including but not limited to uncontrolled growth of organ and
tissue cells resulting in cancers or neoplasia and benign tumors.
Hyperproliferative disorders associated with endothelial cells can
result in diseases of angiogenesis such as angiomas, endometriosis,
obesity, age-related macular degeneration and various
retinopathies, as well as the proliferation of endothelial cells
and smooth muscle cells that cause restenosis as a consequence of
stenting in the treatment of atherosclerosis. Hyperproliferative
disorders involving fibroblasts (for example, fibrogenesis) include
but are not limited to disorders of excessive scarring (for
example, fibrosis) such as age-related macular degeneration,
cardiac remodeling and failure associated with myocardial
infarction, excessive wound healing such as commonly occurs as a
consequence of surgery or injury, keloids, and fibroid tumors and
stenting.
The compositions of the invention are used in methods of
sphingolipid-based therapy. "Therapy" refers to the prevention
and/or treatment of diseases, disorders or physical trauma.
"Cardiovascular therapy" encompasses cardiac therapy as well as the
prevention and/or treatment of other diseases associated with the
cardiovascular system, such as heart disease. The term "heart
disease" encompasses any type of disease, disorder, trauma or
surgical treatment that involves the heart or myocardial tissue. Of
particular interest are heart diseases that relate to hypoxia
and/or ischemia of myocardial tissue and/or heart failure. One type
of heart disease that can result from ischemia is reperfusion
injury, such as can occur when anti-coagulants, thrombolytic
agents, or anti-anginal medications are used in therapy, or when
the cardiac vasculature is surgically opened by angioplasty or by
coronary artery grafting. Another type of heart disease to which
the invention is directed is coronary artery disease (CAD), which
can arise from arteriosclerosis, particularly atherosclerosis, a
common cause of ischemia. CAD has symptoms such as stable or
unstable angina pectoris, and can lead to myocardial infarctions
(MI) and sudden cardiac death. Conditions of particular interest
include, but are not limited to, myocardial ischemia; acute
myocardial infarction (AMI); coronary artery disease (CAD); acute
coronary syndrome (ACS); cardiac cell and tissue damage that may
occur during or as a consequence of pericutaneous revascularization
(coronary angioplasty) with or without stenting; coronary bypass
grafting (CABG) or other surgical or medical procedures or
therapies that may cause ischemic or ischemic/reperfusion damage in
humans; and cardiovascular trauma. The term "heart failure"
encompasses acute myocardial infarction, myocarditis, a
cardiomyopathy, congestive heart failure, septic shock, cardiac
trauma and idiopathic heart failure. The spectrum of ischemic
conditions that result in heart failure is referred to as Acute
Coronary Syndrome (ACS).
The term "cardiotherapeutic agent" refers to an agent that is
therapeutic to diseases and diseases caused by or associated with
cardiac and myocardial diseases and disorders.
"Cerebrovascular therapy" refers to therapy directed to the
prevention and/or treatment of diseases and disorders associated
with cerebral ischemia and/or hypoxia. Of particular interest is
cerebral ischemia and/or hypoxia resulting from global ischemia
resulting from a heart disease, including without limitation heart
failure.
The term "sphingolipid metabolite" refers to a compound from which
a sphingolipid is made, as well as a that results from the
degradation of a particular sphingolipid. In other words, a
"sphingolipid metabolite" is a compound that is involved in the
sphingolipid metabolic pathways. Metabolites include metabolic
precursors and metabolic products. The term "metabolic precursors"
refers to compounds from which sphingolipids are made. Metabolic
precursors of particular interest include but are not limited to
SPC, sphingomyelin, dihydrosphingosine, dihydroceramide, and
3-ketosphinganine. The term "metabolic products" refers to
compounds that result from the degradation of sphingolipids, such
as phosphorylcholine (e.g., phosphocholine, choline phosphate),
fatty acids, including free fatty acids, and hexadecanal (e.g.,
palmitaldehyde).
As used herein, the term "therapeutic" encompasses the fill
spectrum of treatments for a disease or disorder. A "therapeutic"
agent of the invention may act in a manner that is prophylactic or
preventive, including those that incorporate procedures designed to
target individuals that can be identified as being at risk
(pharmacogenetics); or in a manner that is ameliorative or curative
in nature; or may act to slow the rate or extent of the progression
of at least one symptom of a disease or disorder being treated; or
may act to minimize the time required, the occurrence or extent of
any discomfort or pain, or physical limitations associated with
recuperation from a disease, disorder or physical trauma; or may be
used as an adjuvant to other therapies and treatments.
"Treatment" refers to both therapeutic treatment and prophylactic
or preventative measures. Those in need of treatment include those
already with the disorder as well as those in which the disorder is
to be prevented.
The term "combination therapy" refers to a therapeutic regimen that
involves the provision of at least two distinct therapies to
achieve an indicated therapeutic effect. For example, a combination
therapy may involve the administration of two or more chemically
distinct active ingredients, for example, a fast-acting
chemotherapeutic agent and an anti-lipid antibody. Alternatively, a
combination therapy may involve the administration of an anti-lipid
antibody and/or one or more chemotherapeutic agents, alone or
together with the delivery of another treatment, such as radiation
therapy and/or surgery. Further, a combination therapy may involve
administration of an anti-lipid antibody together with one or more
other biological agents (e.g., anti-VEGF, TGF.beta., PDGF, or bFGF
agent), chemotherapeutic agents and another treatment such as
radiation and/or surgery. In the context of the administration of
two or more chemically distinct active ingredients, it is
understood that the active ingredients may be administered as part
of the same composition or as different compositions. When
administered as separate compositions, the compositions comprising
the different active ingredients may be administered at the same or
different times, by the same or different routes, using the same of
different dosing regimens, all as the particular context requires
and as determined by the attending physician. Similarly, when one
or more anti-lipid antibody species, for example, an anti-LPA
antibody, alone or in conjunction with one or more chemotherapeutic
agents are combined with, for example, radiation and/or surgery,
the drug(s) may be delivered before or after surgery or radiation
treatment.
"Monotherapy" refers to a treatment regimen based on the delivery
of one therapeutically effective compound, whether administered as
a single dose or several doses over time.
"Neoplasia" or "cancer" refers to abnormal and uncontrolled cell
growth. A "neoplasm", or tumor or cancer, is an abnormal,
unregulated, and disorganized proliferation of cell growth, and is
generally referred to as cancer. A neoplasm may be benign or
malignant. A neoplasm is malignant, or cancerous, if it has
properties of destructive growth, invasiveness, and metastasis.
Invasiveness refers to the local spread of a neoplasm by
infiltration or destruction of surrounding tissue, typically
breaking through the basal laminas that define the boundaries of
the tissues, thereby often entering the body's circulatory system.
Metastasis typically refers to the dissemination of tumor cells by
lymphatics or blood vessels. Metastasis also refers to the
migration of tumor cells by direct extension through serous
cavities, or subarachnoid or other spaces. Through the process of
metastasis, tumor cell migration to other areas of the body
establishes neoplasms in areas away from the site of initial
appearance.
"Mammal" for purposes of treatment refers to any animal classified
as a mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
Preferably, the mammal is human.
"Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 Daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light-chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light- and heavy-chain variable
domains.
The term "variable" region comprises framework and CDRs (otherwise
known as hypervariables) and refers to the fact that certain
portions of the variable domains differ extensively in sequence
among antibodies and are used in the binding and specificity of
each particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework region (FR). The variable domains
of native heavy and light chains each comprise four FRs (FR1, FR2,
FR3 and FR4, respectively), largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.alpha.-sheet structure. The hypervariable regions in each chain
are held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat, et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), pages 647-669). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
The term "hypervariable region" when used herein refers to the
amino acid residues of an antibody which are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (for
example, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102
(H3) in the heavy chain variable domain; Kabat, et al. (1991),
above) and/or those residues from a "hypervariable loop" (for
example residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the
light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101
(H3) in the heavy chain variable domain; Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). "Framework" or "FR" residues are those
variable domain residues other than the hypervariable region
residues as herein defined.
Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment that contains a complete
antigen-recognition and -binding site. This region consists of a
dimer of one heavy chain and one light chain variable domain in
tight, non-covalent association. It is in this configuration that
the three hypervariable regions of each variable domain interact to
define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six hypervariable regions
confer antigen-binding specificity to the antibody. However, even a
single variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
The Fab fragment also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteine(s) from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of
their heavy chains, immunoglobulins can be assigned to different
classes. Presently there are five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA, and IgA2. The heavy-chain constant domains that correspond to
the different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), antibody fragments, and
binding agents that employ the CDRs (or variant thereof that retain
antigen binding activity) of the parent antibody. Antibodies are
defined herein as retaining at least one desired activity of the
parent antibody. Desired activities can include the ability to bind
the antigen specifically, the ability to inhibit proleration in
vitro, the ability to inhibit angiogenesis in vivo, and the ability
to alter cytokine profile in vitro. "Antibody fragments" comprise a
portion of a full-length antibody, generally the antigen binding or
variable domain thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear
antibodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
for example, the individual antibodies comprising the population
are identical except for possible naturally occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler, et al.,
Nature 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson, et al., Nature 352:624-628 (1991)
and Marks et al., J. Mol. Biol. 222:581-597 (1991), for
example.
The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. No. 4,816,567; and Morrison, et al.,
Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
"Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which
hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones, et al., Nature
321:522-525 (1986); Reichmann, et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) and Hansen,
WO2006105062.
"Single-chain Fv" or "sFv" antibody fragments comprise the V.sub.H
and V.sub.L domains of antibody, wherein these domains are present
in a single polypeptide chain. Generally, the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains that enables the sFv to form the desired structure
for antigen binding. For a review of sFv, see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger, et al., Proc.
Natl. Acad. Sci. USA 90:6444-6448 (1993).
The expression "linear antibodies" when used throughout this
application refers to the antibodies described in Zapata, et al.
Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies
comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) that form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
A "variant" anti-sphingolipid antibody, refers herein to a molecule
which differs in amino acid sequence from a "parent"
anti-sphingolipid antibody amino acid sequence by virtue of
addition, deletion, and/or substitution of one or more amino acid
residue(s) in the parent antibody sequence and retains at least one
desired activity of the parent anti-binding antibody. Desired
activities can include the ability to bind the antigen
specifically, the ability to inhibit proleration in vitro, the
ability to inhibit angiogenesis in vivo, and the ability to alter
cytokine profile in vitro. In one embodiment, the variant comprises
one or more amino acid substitution(s) in one or more hypervariable
region(s) of the parent antibody. For example, the variant may
comprise at least one, e.g. from about one to about ten, and
preferably from about two to about five, substitutions in one or
more hypervariable regions of the parent antibody. Ordinarily, the
variant will have an amino acid sequence having at least 50% amino
acid sequence identity with the parent antibody heavy or light
chain variable domain sequences, more preferably at least 65%, more
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, and most
preferably at least 95% sequence identity. Identity or homology
with respect to this sequence is defined herein as the percentage
of amino acid residues in the candidate sequence that are identical
with the parent antibody residues, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity. None of N-terminal, C-terminal, or internal
extensions, deletions, or insertions into the antibody sequence
shall be construed as affecting sequence identity or homology. The
variant retains the ability to bind a sphingolipid and preferably
has desired activities which are superior to those of the parent
antibody. For example, the variant may have a stronger binding
affinity, enhanced ability to reduce angiogenesis and/or halt tumor
progression. To analyze such desired properties (for example less
immunogenic, longer half-life, enhanced stability, enhanced
potency), one should compare a Fab form of the variant to a Fab
form of the parent antibody or a full length form of the variant to
a full length form of the parent antibody, for example, since it
has been found that the format of the anti-sphingolipid antibody
impacts its activity in the biological activity assays disclosed
herein. The variant antibody of particular interest herein can be
one which displays at least about 5%, preferably at least about
10%, 25%, 59%, or more of at least one desired activity. The
preferred variant is one that has superior biophysical properties
as measured in vitro or superior activities biological as measured
in vitro or in vivo when compared to the parent antibody.
The "parent" antibody herein is one that is encoded by an amino
acid sequence used for the preparation of the variant. Preferably,
the parent antibody has a human framework region and, if present,
has human antibody constant region(s). For example, the parent
antibody may be a humanized or human antibody.
An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
The word "label" when used herein refers to a detectable compound
or composition that is conjugated directly or indirectly to the
antibody. The label may itself be detectable by itself (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition that is detectable.
By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere or upon which the
antibody or other anti-S1P binding reagent can otherwise become
immobilized. Examples of solid phases encompassed herein include
those formed partially or entirely of glass (e.g., controlled pore
glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and silicones. In certain
embodiments, depending on the context, the solid phase can comprise
the well of an assay plate, while in others it is a purification
column (e.g., an affinity chromatography column). This term also
includes a discontinuous solid phase of discrete particles, such as
those described in U.S. Pat. No. 4,275,149.
A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant that is useful for delivery
of a drug (such as the anti-sphingolipid antibodies disclosed
herein and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
nucleic acid molecules being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
As used herein, the expressions "cell", "cell line", and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived there from
without regard for the number of transfers. It is also understood
that all progeny may not be precisely identical in DNA content, due
to deliberate or inadvertent mutations. Mutant progeny that have
the same function or biological activity as screened for in the
originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
A "patentable" composition, process, machine, or article of
manufacture according to the invention means that the subject
matter satisfies all statutory requirements for patentability at
the time the analysis is performed. For example, with regard to
novelty, non-obviousness, or the like, if later investigation
reveals that one or more claims encompass one or more embodiments
that would negate novelty, non-obviousness, etc., the claim(s),
being limited by definition to "patentable" embodiments,
specifically exclude the unpatentable embodiment(s). Also, the
claims appended hereto are to be interpreted both to provide the
broadest reasonable scope, as well as to preserve their validity.
Furthermore, the claims are to be interpreted in a way that (1)
preserves their validity and (2) provides the broadest reasonable
interpretation under the circumstances, if one or more of the
statutory requirements for patentability are amended or if the
standards change for assessing whether a particular statutory
requirement for patentability is satisfied from the time this
application is filed or issues as a patent to a time the validity
of one or more of the appended claims is questioned.
The term "pharmaceutically acceptable salt" refers to salts which
retain the biological effectiveness and properties of the agents
and compounds of this invention and which are not biologically or
otherwise undesirable. In many cases, the agents and compounds of
this invention are capable of forming acid and/or base salts by
virtue of the presence of charged groups, for example, charged
amino and/or carboxyl groups or groups similar thereto.
Pharmaceutically acceptable acid addition salts may be prepared
from inorganic and organic acids, while pharmaceutically acceptable
base addition salts can be prepared from inorganic and organic
bases. For a review of pharmaceutically acceptable salts (see
Berge, et al. (1977) J. Pharm. Sci., vol. 66, 1-19).
A "plurality" means more than one.
The terms "separated", "purified", "isolated", and the like mean
that one or more components of a sample contained in a
sample-holding vessel are or have been physically removed from, or
diluted in the presence of, one or more other sample components
present in the vessel. Sample components that may be removed or
diluted during a separating or purifying step include, chemical
reaction products, unreacted chemicals, proteins, carbohydrates,
lipids, and unbound molecules.
The term "species" is used herein in various contexts, e.g., a
particular species of chemotherapeutic agent. In each context, the
term refers to a population of chemically indistinct molecules of
the sort referred in the particular context.
"Specifically associate" and "specific association" and the like
refer to a specific, non-random interaction between two molecules,
which interaction depends on the presence of structural,
hydrophobic/hydrophilic, and/or electrostatic features that allow
appropriate chemical or molecular interactions between the
molecules.
A "subject" or "patient" refers to an animal in need of treatment
that can be effected by molecules of the invention. Animals that
can be treated in accordance with the invention include
vertebrates, with mammals such as bovine, canine, equine, feline,
ovine, porcine, and primate (including humans and non-human
primates) animals being particularly preferred examples.
A "therapeutically effective amount" (or "effective amount") refers
to an amount of an active ingredient, e.g., an agent according to
the invention, sufficient to effect treatment when administered to
a subject or patient. Accordingly, what constitutes a
therapeutically effective amount of a composition according to the
invention may be readily determined by one of ordinary skill in the
art. In the context of ocular therapy, a "therapeutically effective
amount" is one that produces an objectively measured change in one
or more parameters associated with treatment of the ocular disease
or condition including an increase or decrease in the expression of
one or more genes correlated with the ocular disease or condition,
induction of apoptosis or other cell death pathways, clinical
improvement in symptoms, a decrease in aberrant neovascularization
or in inflammation, etc. Of course, the therapeutically effective
amount will vary depending upon the particular subject and
condition being treated, the weight and age of the subject, the
severity of the disease condition, the particular compound chosen,
the dosing regimen to be followed, timing of administration, the
manner of administration and the like, all of which can readily be
determined by one of ordinary skill in the art. It will be
appreciated that in the context of combination therapy, what
constitutes a therapeutically effective amount of a particular
active ingredient may differ from what constitutes a
therapeutically effective amount of the active ingredient when
administered as a monotherapy (ie., a therapeutic regimen that
employs only one chemical entity as the active ingredient).
The term "treatment" or "treating" of a disease or disorder
includes preventing or protecting against the disease or disorder
(that is, causing the clinical symptoms not to develop); inhibiting
the disease or disorder (i.e., arresting or suppressing the
development of clinical symptoms; and/or relieving the disease or
disorder (i.e., causing the regression of clinical symptoms). As
will be appreciated, it is not always possible to distinguish
between "preventing" and "suppressing" a disease or disorder since
the ultimate inductive event or events may be unknown or latent.
Accordingly, the term "prophylaxis" will be understood to
constitute a type of "treatment" that encompasses both "preventing"
and "suppressing." The term "treatment" thus includes
"prophylaxis".
The term "therapeutic regimen" means any treatment of a disease or
disorder using chemotherapeutic drugs, radiation therapy, surgery,
gene therapy, DNA vaccines and therapy, antisense-based therapies
including siRNA therapy, anti-angiogenic therapy, immunotherapy,
bone marrow transplants, aptamers and other biologics such as
antibodies and antibody variants, receptor decoys and other
protein-based therapeutics.
SUMMARY OF THE INVENTION
This invention concerns patentable humanized anti-sphingolipid
agents, including antibodies and anti-sphingolipid antibody
variants with desirable properties from a therapeutic and/or
diagnostic perspective, including strong binding affinity for
sphingolipids, the ability to bind and neutralize
sphingosine-1-phosphate (S1P), particularly in physiological
contexts (e.g., in living tissue, blood, etc.) and under
physiological conditions, as well as isoforms, variants, isomers,
and related compounds. In particular, the invention is drawn to
antibodies, particularly monoclonal antibodies, more particularly
humanized monoclonal antibodies and variants thereof, directed to
S1P. Such antibodies and variants are preferably included in
pharmaceutical compositions suitable for administration to subjects
in known or suspected to need treatment with such compounds. In
addition to compositions, the invention also provides kits
including such compositions, methods of making such anti-S1P
antibodies and variants, and methods of treatment using such
agents.
In one embodiment, isolated anti-S1P antibody heavy chains and
light chains comprising variable domains of newly identified
preferred sequences, particularly SEQ ID NO: 27 and SEQ ID NO: 35
for heavy chains and SEQ ID NO: 30 and SEQ ID NO: 37 for light
chains, are provided. In another embodiment, anti-S1P agents are
provided that are reactive against sphingosine-1-phosphate (S1P)
under physiological conditions and which comprises at least one CDR
peptide having at least 50% amino acid sequence identity, and up to
and including 100% identity, with the CDR sequences specified
elsewhere herein.
In one embodiment, an anti-sphingolipid antibody according to the
invention has a light chain variable domain comprises hypervariable
complementarity determining regions (CDRs) with the following amino
acid sequences: ITTTDIDDDMN (SEQ ID NO:10; CDRL1), EGNILRP (SEQ ID
NO:11; CDRL2) and LQSDNLPFT (SEQ ID NO:12; CDRL3). Preferably the
heavy chain variable domain comprises CDRs having the amino acid
sequences DHTIH (SEQ ID NO:13; CDRH1), GGFYGSTIWFDF (SEQ ID NO:15;
CDRH3) and CISPRHDITKYNEMFRG (SEQ ID NO:14; CDRH2) or
AISPRHDITKYNEMFRG (SEQ ID NO:31; CDRH2). In particularly preferred
embodiments of the invention, one or more of the CDRs is(are)
grafted into a framework in such a way that the CDRs retain their
ability to bind and neutralize S1P. Without being limited to the
following example, the framework could represent the human sequence
of an antibody light and heavy chains immediately flanking the
CDRs, but could also represent any structure that presents the CDRs
in a way that optimizes the performance characteristics of the
humanized antibody in its binding to the S1P or in other
characteristics that enhance potency, stability, expression,
biological half-life, solubility, immunogenicity,
pharmacodistribution, and shelf-life of the antibody.
Preferably, the three heavy chain hypervariable CDR regions are
provided in a human framework region, e.g., as a contiguous
sequence represented by the following formula:
FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4.
The invention further provides an anti-sphingolipid antibody heavy
chain variable domain comprising the amino acid sequence
represented herein by SEQ ID NO:27. One particularly useful heavy
chain variable domain sequence is that of the humanized antibody
described in Example 12, below, and comprises the heavy chain
variable domain sequence of SEQ ID NO: 32. Such preferred heavy
chain variable domain sequences may be combined with, for example,
a polypeptide comprising the light chain variable domain sequence
represented herein by SEQ ID NO: 33, or with other light chain
variable domain sequences, provided that the resulting molecule
binds a sphingolipid.
In another embodiment, the invention provides a humanized
anti-sphingolipid antibody light chain variable domain comprising
the amino acid sequence represented herein by SEQ ID NO: 17. In one
embodiment, one useful light chain variable domain sequence is that
of the humanized antibody of Example 12, below, and comprises the
light chain variable domain sequence of SEQ ID NO:30 or SEQ ID
NO:37.
In one preferred embodiment, the invention provides a humanized
anti-sphingolipid antibody having a light chain comprising the
amino acid sequence of SEQ ID NO:37 and a heavy chain comprising
the amino acid sequence of SEQ ID NO:35.
The light chain variable domain may comprise hypervariable regions
with the following amino acid sequences: CDRL1 (SEQ ID NO:10),
CDRL2 (SEQ ID NO:11), and CDRL3 (SEQ ID NO:12). Preferably, the
three light chain hypervariable regions are provided in a human
framework region, e.g., as a contiguous sequence represented by the
following formula: FR1-CDRL1-FR2-CDRL2-FR3-CD-RL3-FR4.
The invention also provides variants of parent anti-sphingolipid
antibodies, preferably wherein the parent antibody is a humanized
or human anti-sphingolipid antibody. Such variants bind a
sphingolipid, particularly S1P, and comprise an amino acid
substitution in a hypervariable region of the heavy or light chain
variable domain of the parent anti-sphingolipid antibody. Such a
variant preferably has one or more substitution(s) in one or more
hypervariable region(s) of the anti-sphingolipid antibody.
According to one embodiment, the substitution(s) are in the heavy
chain variable domain of the parent antibody. For example, the
amino acid substitution(s) can be in the CDRH1 and/or CDRH3 of the
heavy chain variable domain. There can be substitutions in both
these hypervariable regions. Such "affinity matured" variants are
demonstrated herein to bind sphingolipid more strongly than the
parent anti-sphingolipid antibody from which they were generated.
For example, an antibody produced by affinity maturation can have a
K.sub.d value that is significantly less than that of the parent
anti-sphingolipid antibody.
A representative example of affinity maturation involves altering
human IgG kappa1 light and heavy chain frameworks into which murine
anti-S1P CDRs were grafted. This resulted in an increased affinity
of the humanized antibody against its target ligand, i.e., S1P. In
other embodiments, one or more of the CDRs could be supported by
amino acid sequences other than human IgG frameworks. Affinity
maturation by altering the amino acid sequence or sequences in the
hypervariable CDR regions can be performed to improve antibody
performance and/or characteristics described above. An example of
this form of affinity maturation is shown in Example 12, below,
where a cysteine residue in a heavy chain CDR was changed by
site-directed mutagenesis to an alanine residue, resulting in a
substantial increase in S1P-binding affinity and stability. In one
such heavy chain variant, the variable region included the amino
acid sequence of SEQ ID NO:27. Such heavy chain variable domain
sequences in CDRH2 can optionally be combined with a light chain
variable domain, for example, a light chain variable comprising the
amino acid sequence of SEQ ID NO:17, or preferably the light chain
variable domain amino acid sequence of SEQ ID NO:30.
Various anti-S1P molecules are contemplated herein. For example,
the anti-S1P agent may be an antibody, an antibody derivative, or a
non-antibody-derived moiety. For example, the anti-S1P agent can be
an antibody, including a full-length antibody (e.g., an antibody
having an intact human Fc region) or an antibody fragment (e.g., an
Fab, Fab', or F(ab').sub.2 molecule), a chimeric antibody, a
humanized antibody, a human antibody, or an affinity matured
antibody. Without limiting the invention, such anti-S1P agents can
be produced to improve or otherwise alter antibody stability,
half-life, potency, pharmacodistribution, and/or immunogenicity.
For example, a humanized Fc domain could be altered in its amino
acid composition to improve its immunogenicity or other performance
characteristics.
In other embodiments, the anti-S1P agent can be conjugated to a
moiety such as a polymer, a radionuclide, a chemotherapeutic agent,
and a detection agent.
In certain preferred embodiments, the anti-S1P agent is formulated
with a carrier such as a pharmaceutically acceptable carrier. In
one embodiment, the anti-S1P agent is combined with a second agent
such as an antibody, an antibody fragment, an antibody derivative,
an antibody variant, a therapeutic agent other than an anti-S1P
agent, or an agent that can bind a molecule other than S1P.
The instant invention also provides isolated nucleic acid molecules
that encode the various components of antibodies, antibody
variants, and fragments according to the invention, including
various heavy and light chain sequences and CDRs. Vectors and host
cells containing these nucleic acid molecules are also provided.
Further provided are isolated polypeptides comprising one or more
of the preferred amino acid sequences, such as CDR sequences or
antibody light and/or heavy chain sequences.
In preferred embodiments of the invention, isolated antibody
molecules are provided that contain precisely defined CDR sequences
in each heavy chain and each light chain. In one such embodiment,
the isolated antibody molecule is a humanized antibody
molecule.
Multivalent binding molecules having a ligand binding element that
is reactive with S1P and that contain one or more of the preferred
CDR sequences are also provided. These multivalent molecues may
contain at least one, and up to 10,000 or more, ligand binding
elements that are reactive with S1P. Ligand binding elements
reactive a different ligand can also be included, if desired, as
can different ligand binding element species each reactive with S1P
but differing from other S1P binding elements in one or more
characteristics (e.g., molecular structure, binding affinity,
etc).
Also provided are methods of treating or preventing diseases or
disorders correlated with aberrant levels, particularly elevated
levels, of S1P. In general, such methods comprise administering to
a subject, such as a human, in need of such treatment one of the
anti-S1P compositions of the invention. Diseases or disorders
amenable to treatment by such methods include cancer, inflammatory
disorders, cerebrovascular diseases, cardiovascular diseases,
ocular disorders, diseases and disorders associated with excessive
fibrogenesis, and diseases or disorders associated with pathologic
angiogenesis. Anti-S1P compositions can also be administered in
combination with another therapeutic agent or therapeutic
regimen.
A related aspect concerns methods of reducing toxicity of a
therapeutic regimen for treatment or prevention of a
hyperproliferative disorder. Such methods comprise administering to
a subject suffering from a hyperproliferative disorder an effective
amount of an agent (or a plurality of different agent species)
according to the invention before, during, or after administration
of a therapeutic regimen intended to treat or prevent the
hyperproliferative disorder. In a preferred embodiment, the
antibody and the therapeutic regimen have additive effects, and
addition of the antibody to the therapeutic regimen may allow
reduction of the dosage of the therapeutic regimen, thus reducing
treatment-associated toxicity.
Yet another aspect of the invention concerns diagnostic uses for
the anti-S1P agents of the invention. In one diagnostic
application, the invention provides methods for determining the
presence in a sample of a target sphingolipid. In general, such
methods are performed by exposing a sample (such as a bodily fluid
or tissue biopsy sample) suspected of containing a particular
sphingolipid (i.e., the "target" sphingolipid) to an anti-S1P agent
such as anti-sphingolipid antibody of the invention and determining
whether an aberrant level (i.e., a level associated or correlated
with a disease, condition, or disorder) of the target sphingolipid
(e.g., S1P) exists in the sample. For certain of these
applications, kits containing the antibody and instructions for its
use are provided.
Still another aspect of the invention concerns methods of making an
anti-S1P agent. Preferred examples of such agents include
antibodies, antibody variants, and antibody derivatives (e.g.,
antibody fragments). In preferred embodiments, particularly those
that concern anti-S1P agents that comprise on or more polypetides,
biological production systems such as cell lines are preferred. Of
course, synthetic chemistry methods can also be employed.
These and other aspects and embodiments of the invention are
discussed in greater detail in the sections that follow. The
foregoing and other aspects of the invention will become more
apparent from the following detailed description, accompanying
drawings, and the claims. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, suitable methods and materials
are described below. In addition, the materials, methods, and
examples below are illustrative only and not intended to be
limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
A brief summary of each of the figures is provided below.
FIG. 1. FIG. 1 has two panels, A and B. Panel A graphically
illustrates the results of a competitive ELISA for S1P, SPH, LPA,
SPC, and other structurally similar biolipids competing for a
biotin-conjugated anti-S1P monoclonal antibody. These results
indicate that the antibody is specific and sensitive for S1P and
does not recognize structurally similar bioactive lipids. As
described in Example 1, below, bound antibody was detected by a
second antibody, specific for the mouse or human IgG, conjugated
with HRP. Chromogenic reactions were measured and reported as
optical density (OD). The concentration of lipids used for the
competition is indicated on the X-axis. No interaction of the
secondary antibody with S1P coated matrix alone could be detected
(data not shown). Panel B shows the structures of the bioactive
lipids similar to S1P that are listed in Panel A.
FIG. 2. This figure shows the binding properties of several
chimeric and recombinant humanized anti-S1P antibody variants. The
binding to S1P for a chimeric antibody (pATH10+pATH50) was compared
in an ELISA binding assay to two versions of the humanized anti-S1P
monoclonal antibody (pATH201+pATH308) and (pATH201+pATH309).
pATH308 is the humanized light chain with five murine backmutations
and pATH309 is the humanized light chain with three backmutations
in the framework region. The humanized heavy chain (pATH201)
contains only one murine backmutation in the framework region.
FIG. 3 is a graph showing that SPHINGOMAB is highly specific for
S1P. The graph, the data for which were generated using a
competitive ELISA, demonstrates SPHINGOMAB's specificity for S1P as
compared to other bioactive lipids. SPHINGOMAB demonstrated no
cross-reactivity to sphingosine (SPH), the immediate metabolic
precursor of S1P or lysophosphatidic acid (LPA), an important
extracellular signaling molecule that is structurally and
functionally similar to S1P. SPHINGOMAB did not recognize other
structurally similar lipids and metabolites, including
ceramide-1-phosphate (C1P), dihydrosphingosine (DH-SPH),
phosphatidylserine (PS), phosphatidyl ethanolamine (PE), or
sphingomyelin (SM). SPHINGOMAB did cross react with
dihydrosphingosine-1-phosphate (DH-S1P) and, to a lesser extent,
sphingosylphosphoryl choline (SPC). The affinity (Kd) of SPHINGOMAB
for S1P is less than 100 pM, much higher than most therapeutic
antibodies, particularly other molecular sponges.
FIG. 4. FIG. 4 has two parts, A and B. The experiments giving rise
to the data represented in this Figure are detailed in Example 4,
below. Briefly, these data show that SPHINGOMAB reduced CNV and
scar formation in ocular lesions. Mice were treated with SPHINGOMAB
or an isotype-matched non-specific monoclonal antibody. CNV lesions
were induced by laser rupture of Bruchs membrane. Shown are graphs
and representative images of lesions from each treatment group
stained with rhodamine-conjugated R. communis agglutinin I for
vascularization (A) or Masson's Trichrome for collagen scar
formation (B). FIG. 4A shows that in a murine CNV lesion formation
model SPHINGOMAB dramatically attenuates choroidal
neovascularization 14 and 28 days after laser-induced rupture of
Bruch's membranes. FIG. 4B shows that SPHINGOMAB significantly
reduces fibrosis associated with CNV lesion formation 28 days after
laser-induced rupture of Bruchs's membrane.
FIG. 5. FIG. 5 has two panels, A and B. In panel A, S1P is shown to
promote neovascularization through induction of HUVECs tube
formation and migration, which is reduced by SPHINGOMAB. Panel 5A
shows four micrographs of HUVECs seeded on Matrigel and incubated
for 6 hr. to evaluate tube formation. Panel 5B shows data for
HUVECs that were treated with 1 .mu.M S1P.+-.SPHINGOMAB (1
.mu.g/ml) for 6 hr. in a Matrigel invasion chamber. The number of
cells that migrated to the Matrigel membrane were counted in five
independent fields.
FIG. 6. FIG. 6 contains several photographs (A) and graphs (B and
C) for experiments described in Example, 6, below, which were
performed using SPHINGOMAB. SPHINGOMAB neutralizes S1P-, VEGF- and
bFGF-induced neovascularization. FIG. 6A shows photos of several
representative FITC-stained blood vessels from sections of Matrigel
plugs.+-.the indicated growth factors. FIG. 6B shows that S1P
stimulates endothelial cell (EC) infiltration. FIG. 6C represents
the quantification of relative fluorescence from Matrigel plugs
stimulated with VEGF or bFGF as an indicator of neovascularization.
The effects of S1P, VEGF, and bFGF were inhibited when mice were
systemically treated with 1 or 25 mg/kg of SPHINGOMAB.
FIG. 7. FIG. 7 shows 5 graphs, labeled A-E, and two photos. This
data was generated using the anti-S1P monoclonal antibody
SPHINGOMAB. See Example 7, below, for experimental details.
Briefly, these data show that SPHINGOMAB neutralizes S1P-stimulated
scar formation. In these experiments, fibroblasts were
serum-starved and then treated with 0, 0.1, 0.5, or 1 .mu.M S1 +/-1
.mu.g/mL SPHINGOMAB for 12-24 hr. The data show S1P-stimulated
Swiss 3T3 fibroblast proliferation, as measured by 3H-thymidine
incorporation (A), murine cardiac fibroblast migration in a scratch
assay (B), collagen gene expression (relative fluorescence) in
isolated cardiac fibroblasts from transgenic mice expressing
collagen-GFP (C), and WI-38 cell differentiation into
myofibroblasts as measured by decreased cellular proliferation and
increased .alpha.-SMA expression (D). SPHINGOMAB neutralized each
of these S1P effects. SPHINGOMAB reduced perivascular fibrosis in
vivo in a murine model of a permanent myocardial infarction
(E).
FIG. 8. FIG. 8 has three panels, 8A, 8B, and 8C. These data show
that S1P promotes transformation of ocular epithelial cells and
fibroblasts into contractile, scar tissue-producing myofibroblasts.
As described in Example 8, below, the effects of S1P on
myofibroblast transformation of several human ocular cell lines
were examined. S1P was found to stimulate production of
.alpha.-Smooth muscle actin (.alpha.-SMA; a myofibroblast marker)
in human retinal pigmented epithelial cells (FIG. 8A) and human
conjunctiva fibroblasts (FIG. 8B). These data demonstrate, for the
first time, that S1P is among the factors that promote
transformation of ocular epithelial cells and fibroblasts into
contractile, scar tissue-producing myofibroblasts. The effects of
S1P on expression of plasminogen activator inhibitor (PAI-1) in
human conjunctiva fibroblasts were also examined. Increased PAI-1
expression correlates with a decrease in the proteolytic
degradation of connective tissue and is upregulated in association
with several fibrotic diseases that involve increased scarring. As
shown in FIG. 8C, S1P stimulates the PAI-1 expression in a
dose-dependent manner.
FIG. 9. FIG. 9 shows two bar graphs, A and B, showing experimental
data generated using an anti-S1P monoclonal antibody called
SPHINGOMAB. SPHINGOMAB reduced immune-cell wound infiltration in
vivo. Mice were subjected to MI, treated with saline or 25mg/kg
SPHINGOMAB 48 hr. after surgery and then sacrificed on day 4.
SPHINGOMAB reduced macrophage (A) and mast cell (B) infiltration
into the wound. Data are represented as fold decrease of
saline-treated values.
FIG. 10. FIG. 10 has two panels, 10A and 10B. Each panel shows a
map of a cloning vector for expression of murine anti-S1P
monoclonal antibody VL and V.sub.H domains. FIG. 10A is a map of a
pKN100 vector for the cloning of the VL domain. FIG. 10A is a map
of a pG1D200 vector for the cloning of the VH domain.
FIG. 11. FIG. 11 presents data showing the binding properties of
several murine, chimeric, and recombinant humanized anti-S1P
antibodies. The binding to S1P for the mouse (muMA1P; curve
generated from square data points) and chimeric (chMAb S1P; curve
generated from upright triangular data points) antibodies were
compared in an ELISA binding assay to the first version of the
humanized antibody (pATH200+pATH300; curve generated from inverted
triangular data points).
FIG. 12. FIG. 12 has two panels, A and B, that show data from in
vitro cell assays performed using several humanized monoclonal
antibody variants. Panel A shows the humanized mAb is able to
prevent S1P from protecting SKOV3 cells from Taxol-induced
apoptosis. As described in Example 16, below, SKOV3 cells were
treated for 48 hr. with 500 nM Taxol (Tax) in the presence or
absence of 500 nM S1P with huMAbHCLC.sub.3 (309), huMAbHCLC.sub.5
(308), muMAb S1P (muMAb), or non-specific IgG1 (NS) at a
concentration of 1 .mu.g/mL. Values represent means.+-.SEM (n=3)
with triplicates run for each data point. "NT" means not treated,
and "Veh" stands for vehicle only. Panel B shows IL-8 secretion in
ovarian cancer (OVCAR3) cells treated with S1P and one of several
different anti-S1P monoclonal antibodies or a control monoclonal
antibody. In the experiments described in detail in Example 16,
below, 100,000 OVCAR3 cells/well were starved overnight and 1 uM
S1P was added to the culture media alone or pre-incubated with 1
ug/ml of non-specific antibody (NS), pATH201+pATH309 (LC3),
pATH201+pATH308 (LC5), pATH207+pATH309 (cysLC3), pATH207+pATH308
(cysLC5), and 0.1 ug/ml (M0.1), 1 ug/ml (M1) or 10 ug/ml (M10) of
anti-S1P murine antibody. After 22 hours of incubation, cell
supernatants were collected and IL-8 secretion was measured by
ELISA using an R&D Systems Quantikine human CXCL8/IL-8 kit. In
the figure "NT" refers to non-treated cells.
FIG. 13. FIG. 13 shows the in vivo efficacy of several human
monoclonal antibody variants as compared to a mouse anti-S1P
monoclonal antibody and controls in a CNV animal model. As
described in Example 17, below, in these experiments mice were
administered with 0.5 ug twice (day 0 and day 6) of a murine (Mu)
anti-S1P monoclonal antibody, several humanized anti-S1P monoclonal
antibody variants (i.e., variants LC3, LC5, HCcysLC3, and
HCcysLC5), or a non-specific monoclonal antibody (NS) by
intravitreal administration and then subjected to laser rupture of
the Bruch's membrane. Mice were sacrificed 14 days post-laser
surgery. Sclera-RPE-choroid complexes were dissected and stained
with a Rhodamine-conjugated R. communis agglutinin I antibody. CNV
lesion volumes are represented as the means.+-.SEM.
DETAILED DESCRIPTION OF THE INVENTION
1. Compounds.
The present invention describes certain anti-S1P agents,
particularly those that are immune-derived moieties, including
antibodies, which are specifically reactive with the bioactive
lipid S1P; in other words, the bioactive lipid to which the
anti-S1P agent reacts is S1P.
Antibody molecules or immunoglobulins are large glycoprotein
molecules with a molecular weight of approximately 150 kDa, usually
composed of two different kinds of polypeptide chain. One
polypeptide chain, termed the "heavy" chain (H) is approximately 50
kDa. The other polypeptide, termed the "light" chain (L), is
approximately 25 kDa. Each immunoglobulin molecule usually consists
of two heavy chains and two light chains. The two heavy chains are
linked to each other by disulfide bonds, the number of which varies
between the heavy chains of different immunoglobulin isotypes. Each
light chain is linked to a heavy chain by one covalent disulfide
bond. In any given naturally occurring antibody molecule, the two
heavy chains and the two light chains are identical, harboring two
identical antigen-binding sites, and are thus said to be divalent,
i.e., having the capacity to bind simultaneously to two identical
molecules.
The "light" chains of antibody molecules from any vertebrate
species can be assigned to one of two clearly distinct types, kappa
(k) and lambda (l), based on the amino acid sequences of their
constant domains. The ratio of the two types of light chain varies
from species to species. As a way of example, the average k to I
ratio is 20:1 in mice, whereas in humans it is 2:1 and in cattle it
is 1:20.
The "heavy" chains of antibody molecules from any vertebrate
species can be assigned to one of five clearly distinct types,
called isotypes, based on the amino acid sequences of their
constant domains. Some isotypes have several subtypes. The five
major classes of immunoglobulin are immunoglobulin M (IgM),
immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A
(IgA), and immunoglobulin E (IgE). IgG is the most abundant isotype
and has several subclasses (IgG1, 2, 3, and 4 in humans). The Fc
fragment and hinge regions differ in antibodies of different
isotypes, thus determining their functional properties. However,
the overall organization of the domains is similar in all
isotypes.
The term "variable region" refers to the N-terminal portion of the
antibody molecule or a fragment thereof. In general, each of the
four chains has a variable (V) region in its amino terminal
portion, which contributes to the antigen-binding site, and a
constant (C) region, which determines the isotype. The light chains
are bound to the heavy chains by many noncovalent interactions and
by disulfide bonds and the V regions of the heavy and light chains
pair in each arm of antibody molecule to generate two identical
antigen-binding sites. Some amino acid residues are believed to
form an interface between the light- and heavy-chain variable
domains [see Kabat, et al. (1991), Sequences of Proteins of
Immunological Interest, Fifth Edition, National Institute of
Health, Bethesda, Md. and Clothia et al. (1985), J. Mol. Biol, vol
186: 651].
Of note, variability is not uniformly distributed throughout the
variable domains of antibodies, but is concentrated in three
segments called "complementarity-determining regions" (CDRs) or
"hypervariable regions" both in the light-chain and the heavy-chain
variable domains. The more highly conserved portions of variable
domains are called the "framework region" (FR). The variable
domains of native heavy and light chains each comprise four FR
regions connected by three CDRs. The CDRs in each chain are held
together in close proximity by the FR regions and, with the CDRs
from the other chains, form the antigen-binding site of antibodies
[see Kabat, et al. (1991), Sequences of Proteins of Immunological
Interest, Fifth Edition, National Institute of Health, Bethesda,
Md.]. Collectively, the 6 CDRs contribute to the binding properties
of the antibody molecule for the antigen. However, even a single
variable domain (or half of an Fv, comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen [see Pluckthun (1994), in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315].
The term "constant domain" refers to the C-terminal region of an
antibody heavy or light chain. Generally, the constant domains are
not directly involved in the binding properties of an antibody
molecule to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity. Here, "effector functions" refer to the
different physiological effects of antibodies (e.g., opsonization,
cell lysis, mast cell, basophil and eosinophil degranulation, and
other processes) mediated by the recruitment of immune cells by the
molecular interaction between the Fc domain and proteins of the
immune system. The isotype of the heavy chain determines the
functional properties of the antibody. Their distinctive functional
properties are conferred by the carboxy-terminal portions of the
heavy chains, where they are not associated with light chains.
As used herein, "antibody fragment" refers to a portion of an
intact antibody that includes the antigen binding site or variable
regions of an intact antibody, wherein the portion can be free of
the constant heavy chain domains (e.g., CH2, CH3, and CH4) of the
Fc region of the intact antibody. Alternatively, portions of the
constant heavy chain domains (e.g., CH2, CH3, and CH4) can be
included in the "antibody fragment". Examples of antibody fragments
are those that retain antigen-binding and include Fab, Fab',
F(ab')2, Fd, and Fv fragments; diabodies; triabodies; single-chain
antibody molecules (sc-Fv); minibodies, nanobodies, and
multispecific antibodies formed from antibody fragments. By way of
example, a Fab fragment also contains the constant domain of a
light chain and the first constant domain (CH1) of a heavy
chain.
The term "variant" refers to an amino acid sequence which differs
from the native amino acid sequence of an antibody by at least one
amino acid residue or modification. A native or parent or wild-type
amino acid sequence refers to the amino acid sequence of an
antibody found in nature. "Variant" of the antibody molecule
includes, but is not limited to, changes within a variable region
or a constant region of a light chain and/or a heavy chain,
including the hypervariable or CDR region, the Fc region, the Fab
region, the CH1 domain, the CH2 domain, the CH3 domain, and the
hinge region.
The term "specific" refers to the selective binding of an antibody
to its target epitope. Antibody molecules can be tested for
specificity of binding by comparing binding of the antibody to the
desired antigen to binding of the antibody to unrelated antigen or
analogue antigen or antigen mixture under a given set of
conditions. Preferably, an antibody according to the invention will
lack significant binding to unrelated antigens, or even analogs of
the target antigen. Here, the term "antigen" refers to a molecule
that is recognized and bound by an antibody molecule or
immune-derived moiety that binds to the antigen. The specific
portion of an antigen that is bound by an antibody is termed the
"epitope." A "hapten" refers to a small molecule that can, under
most circumstances, elicit an immune response (i.e., act as an
antigen) only when attached to a carrier molecule, for example, a
protein, polyethylene glycol (PEG), colloidal gold, silicone beads,
and the like. The carrier may be one that also does not elicit an
immune response by itself.
The term "antibody" is used in the broadest sense, and encompasses
monoclonal, polyclonal, multispecific (e.g., bispecific, wherein
each arm of the antibody is reactive with a different epitope or
the same or different antigen), minibody, heteroconjugate, diabody,
triabody, chimeric, and synthetic antibodies, as well as antibody
fragments that specifically bind an antigen with a desired binding
property and/or biological activity.
The term "monoclonal antibody" (mAb) refers to an antibody, or
population of like antibodies, obtained from a population of
substantially homogeneous antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, monoclonal antibodies can be made by the hybridoma method
first described by Kohler and Milstein (1975), Nature, vol 256:
495-497, or by recombinant DNA methods.
The term "chimeric" antibody (or immunoglobulin) refers to a
molecule comprising a heavy and/or light chain which is identical
with or homologous to corresponding sequences in antibodies derived
from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical
with or homologous to corresponding sequences in antibodies derived
from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity [Cabilly et al. (1984),
infra; Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851].
The term "humanized antibody" refers to forms of antibodies that
contain sequences from non-human (eg, murine) antibodies as well as
human antibodies. A humanized antibody can include conservative
amino acid substitutions or non-natural residues from the same or
different species that do not significantly alter its binding
and/or biologic activity. Such antibodies are chimeric antibodies
that contain minimal sequence derived from non-human
immunoglobulins. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, camel, bovine, goat, or rabbit having
the desired properties. Furthermore, humanized antibodies can
comprise residues that are found neither in the recipient antibody
nor in the imported CDR or framework sequences. These modifications
are made to further refine and maximize antibody performance. Thus,
in general, a humanized antibody will comprise all of at least one,
and in one aspect two, variable domains, in which all or all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), or that of a human
immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;
Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S.
Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1;
Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,
European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;
Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al.,
European Patent Application No. 0,519,596 A1; Queen et al. (1989)
Proc. Nat'l Acad. Sci. USA, vol 86:10029-10033).
The term "bispecific antibody" can refer to an antibody, or a
monoclonal antibody, having binding properties for at least two
different epitopes. In one embodiment, the epitopes are from the
same antigen. In another embodiment, the epitopes are from two
different antigens. Methods for making bispecific antibodies are
known in the art. For example, bispecific antibodies can be
produced recombinantly using the co-expression of two
immunoglobulin heavy chain/light chain pairs. Alternatively,
bispecific antibodies can be prepared using chemical linkage.
Bispecific antibodies include bispecific antibody fragments.
The term "heteroconjugate antibody" can refer to two covalently
joined antibodies. Such antibodies can be prepared using known
methods in synthetic protein chemistry, including using
crosslinking agents. As used herein, the term "conjugate" refers to
molecules formed by the covalent attachment of one or more antibody
fragment(s) or binding moieties to one or more polymer
molecule(s).
The term "biologically active" refers to an antibody or antibody
fragment that is capable of binding the desired epitope and in some
way exerting a biologic effect. Biological effects include, but are
not limited to, the modulation of a growth signal, the modulation
of an anti-apoptotic signal, the modulation of an apoptotic signal,
the modulation of the effector function cascade, and modulation of
other ligand interactions.
The term "recombinant DNA" refers to nucleic acids and gene
products expressed therefrom that have been engineered, created, or
modified by man. "Recombinant" polypeptides or proteins are
polypeptides or proteins produced by recombinant DNA techniques,
for example, from cells transformed by an exogenous DNA construct
encoding the desired polypeptide or protein. "Synthetic"
polypeptides or proteins are those prepared by chemical
synthesis.
The term "expression cassette" refers to a nucleotide molecule
capable of affecting expression of a structural gene (i.e., a
protein coding sequence, such as an antibody of the invention) in a
host compatible with such sequences. Expression cassettes include
at least a promoter operably linked with the polypeptide-coding
sequence, and, optionally, with other sequences, e.g.,
transcription termination signals. Additional regulatory elements
necessary or helpful in effecting expression may also be used,
e.g., enhancers. Thus, expression cassettes include plasmids,
expression vectors, recombinant viruses, any form of recombinant
"naked DNA" vector, and the like.
Sources of antibody are not limited to those exemplified herein
(e.g., murine and humanized murine antibody). Antibodies may be
raised in many species including mammalian species (for example,
mouse, rat, camel, bovine, goat, horse, guinea pig, hamster, sheep
and rabbit) and birds (duck, chicken). Antibodies raised may derive
from a different species from the animal in which they are raised.
For example, the XenoMouse.TM. (Abgenix, Inc., Fremont Calif.)
produces fully human monoclonal antibodies. For certain purposes,
native human antibodies, such as autoantibodies to S1P isolated
from individuals who may show a titer of such S1P autoantibody may
be used. Alternatively, a human antibody sequence library may be
used to generate antibodies comprising a human sequence.
2. Applications.
The invention is drawn to compositions and methods for treating or
preventing certain diseases and conditions, using one or more
therapeutic agents that alter the activity or concentration of one
or more undesired bioactive lipids, or precursors or metabolites
thereof. The therapeutic methods and compositions of the invention
act by changing the effective concentration, i.e., the absolute,
relative, effective and/or available concentration and/or
activities, of certain undesired bioactive lipids. Lowering the
effective concentration of the bioactive lipid may be said to
"neutralize" the target lipid or its undesired effects, including
downstream effects. Here, "undesired" refers to a bioactive lipid
that is unwanted due to its involvement in a disease process, for
example, as a signaling molecule, or to an unwanted amount of a
bioactive lipid which contributes to disease when present in
excess.
Without wishing to be bound by any particular theory, it is
believed that inappropriate concentrations of S1P and/or its
metabolites or downstream effectors, may cause or contribute to the
development of various diseases and disorders. As such, the
compositions and methods can be used to treat these diseases and
disorders, particularly by decreasing the effective in vivo
concentration of a particular target lipid, for example, S1P or its
variants. In particular, it is believed that the compositions and
methods of the invention are useful in treating diseases
characterized, at least in part, by aberrant neovascularization,
angiogenesis, fibrogenesis, fibrosis, scarring, inflammation, and
immune response.
Examples of several classes of diseases that may be treated in
accordance with the invention are described below. It will be
appreciated that many disease and conditions are characterized, at
least in part, by multiple pathological processes (for example,
both pathological neovascularization and scarring) and that the
classifications provided herein are for descriptive convenience and
do not limit the invention.
S1P and Hyperproliferative Disorders
One aspect of the invention concerns methods for treating a
hyperproliferative disorder. These methods comprise administering
to a mammal (e.g., a bovine, canine, equine, ovine, or porcine
animal, particularly a human) known or suspected to suffer from an
S1P-associated hyperproliferative disorder a therapeutically
effective amount of a composition comprising an agent that
interferes with S1P activity, preferably in a pharmaceutically or
veterinarily acceptable carrier, as the intended application may
require. S1P-associated hyperproliferative disorders include
neoplasias, disorder associated with endothelial cell
proliferation, and disorders associated with fibrogenesis. Most
often, the neoplasia will be a cancer. Typical disorders associated
with endothelial cell proliferation are angiogenesis-dependent
disorders, for example, cancers caused by a solid tumors,
hematological tumors, and age-related macular degeneration.
Disorders associated with fibrogenesis include those than involve
aberrant cardiac remodeling, such as cardiac failure.
There are many known hyperproliferative disorders, in which cells
of various tissues and organs exhibit aberrant patterns of growth,
proliferation, migration, signaling, senescence, and death. While a
number of treatments have been developed to address some of these
diseases, many still remain largely untreatable with existing
technologies, while in other cases, while treatments are available,
they are frequently less than optimal and are seldom curative.
Cancer represents perhaps the most widely recognized class of
hyperproliferative disorders. Cancers are a devastating class of
diseases, and together, they have a mortality rate second only to
cardiovascular disease. Many cancers are not fully understood on a
molecular level. As a result, cancer is a major focus of research
and development programs for both the United States government and
pharmaceutical companies. The result has been an unprecedented
R&D effort and the production of many valuable therapeutic
agents to help in the fight against cancer.
Unfortunately the enormous amount of cancer research has not been
enough to overcome the significant damage caused by cancer. There
are still over one million new cases of cancer diagnosed annually
and over five hundred thousand deaths in the United States alone.
This is a dramatic demonstration that even though an enormous
effort has been put forth to discover new therapeutics for cancer,
effective therapeutic agents to combat the disease remain
elusive.
Cancer is now primarily treated with one or a combination of three
types of therapies, surgery, radiation, and chemotherapy. Surgery
involves the bulk removal of diseased tissue. While surgery is
sometimes effective in removing tumors located at certain sites,
for example, in the breast, colon, and skin, it cannot be used in
the treatment of tumors located in other areas, such as the
backbone, nor in the treatment of disseminated neoplastic
conditions such as leukemia. Radiation therapy involves the
exposure of living tissue to ionizing radiation causing death or
damage to the exposed cells. Side effects from radiation therapy
may be acute and temporary, while others may be irreversible.
Chemotherapy involves the disruption of cell replication or cell
metabolism.
Further insult is that current therapeutic agents usually involve
significant drawbacks for the patient in the form of toxicity and
severe side effects. Therefore, many groups have recently begun to
look for new approaches to fighting the war against cancer. These
new so-called "innovative therapies" include gene therapy and
therapeutic proteins such as monoclonal antibodies.
The first monoclonal used in the clinic for the treatment of cancer
was Rituxan (rituximab) which was launched in 1997, and has
demonstrated the utility of biospecific monoclonal antibodies as
therapeutic agents. Thus, not surprisingly, sixteen other
monoclonal antibodies have since been approved for use in the
clinic, including six that are prescribed for cancer. The success
of these products, as well as the reduced cost and time to develop
monoclonal antibodies as compared with small molecules has made
monoclonal antibody therapeutics the second largest category of
drug candidates behind small molecules. Further, the exquisite
specificity of antibodies as compared to small molecule
therapeutics has proven to be a major advantage both in terms of
efficacy and toxicity. For cancer alone there are currently more
than 270 industry antibody R&D projects with more than 50
companies involved in developing new cancer antibody therapeutics.
Consequently, monoclonal antibodies are poised to become a major
player in the treatment of cancer and they are estimated to capture
an increasing share of the cancer therapeutic market.
The identification of extracellular mediators that promote tumor
growth and survival is a critical step in discovering therapeutic
interventions that will reduce the morbidity and mortality of
cancer. As described below, sphingosine-1-phosphate (S1P), a key
component of sphingolipid signaling cascade, is considered to be a
pleiotropic, tumorigenic growth factor. S1P promotes tumor growth
by stimulating cell proliferation, cell survival, and metastasis.
S1P also promotes tumor angiogenesis by supporting the migration
and survival of endothelial cells as they form new vessels within
tumors. Taken together, S1P initiates a proliferative,
pro-angiogenic, and anti-apoptotic sequence of events contributing
to cancer progression. Thus, therapies that modulate, and, in
particular, reduce S1P levels in vivo will be effective in the
treatment of cancer.
Research has demonstrated that sphingosine kinase (SPHK) is a
recently validated oncogene that produces an extracellular
sphingolipid signaling molecule, sphingosine-1-phosphate (S1P) that
promotes tumor growth. Tumor growth is promoted both directly and
indirectly by S1P's growth factor actions related to tumor cell
proliferation and metastasis, as well as S1P's pro-angiogenic
effects. The applicant has produced a biospecific monoclonal
anti-S1P antibody (anti-S1P mAb) that could be used as a
therapeutic molecular sponge to selectively absorb S1P, thus
lowering extracellular concentrations of this tumor growth factor
with the anticipated reduction in tumor volume and metastatic
potential as well as simultaneously blocking new blood vessel
formation that would, otherwise, feed the growing tumor. The
anticipated success of the molecular absorption concept will
represent an innovative approach to the treatment of cancer. As the
paragraphs below will demonstrate, the applicant has developed a
mAb against an important tumor growth factor,
sphingosine-1-phosphate (S1P). The applicant believes that this
antibody can be effective in reduced the proliferation, metastatic
potential and angiogenesis associated with many cancer types, and
therefore, cancer in general as well as the tumor angiogenesis that
accompanies the disease.
The neutral form of sphingomyelinase (nSMase) is a key early
component of the sphingolipid signaling pathway (Chatterjee, Adv.
Lipid Res. 26: 25-46, 1993; Liu, Obein, and Hannun, Semin. Cell
Dev. Biol. 8: 311-322, 1997) nSMase is only one of at least five
classes of SMase that have been identified, including the alkaline,
the acidic, the acidic zinc-dependent, the neutral
magnesium-dependent, and the neutral magnesium-independent (Liu,
Obein, and Hannun, Semin. Cell Dev. Biol. 8: 311-322, 1997). The
nSMase class is commonly associated with surface membranes (Das,
Cook, and Spence, Biochim Biophys Acta 777: 339-342, 1984;
Dobrowsky, Cell Signal 12: 81-90., 2000) and can be activated by a
variety of stimuli to cause apoptosis, such as the pro-inflammatory
cytokine, tumor necrosis factor alpha (TNF.alpha.) (Segui, et al.,
J. Clin. Invest. 108: 143-151, 2001), T cell receptor (Tonnetti, et
al., J. Exp. Med. 189: 1581-1589, 1999), ionizing radiation
(Haimovitz-Friedman, et al., J. Exp. Med. 180: 525-535, 1994) and
the anthracycline anti-neoplastic agents (Andrieu-Abadie, et al.,
FASEB J. 13: 1501-1510, 1999). Tumor necrosis factor alpha
(TNF.alpha.) is a well-known activator of nSMase (Adam, et al., J.
Bio Chem 271: 14617-14622, 1996; Dressler, Mathias, and Kolesnick,
Science 255: 1715-1718, 1992; Kim, et al., J. Biol. Chem. 266:1:
484-489, 1991; Kronke, Chem Phys Lipids 102: 157-66., 1999; Yanaga
and Watson, FEBS Letters 314: 297-300, 1992), CER production
(Kronke, Chem Phys Lipids 102: 157-66., 1999) and apoptosis (Rath
and Aggarwal, J. Clin. Immuno. 19: 350-364, 1999; Robaye, et al.,
Am J Pathol 138: 447-453, 1991; Takeda et al., Int. Immunol. 5:
691-694, 1993) in many cell types, including cancer cell lines
(Andrieu-Abadie, et al., FASEB J. 13: 1501-1510, 1999; Hannun and
Obein, Trends in Biol. Sci. 20: 72-76, 1995; Kolesnick, trends
Biochem Sci 24: 224-5, 1999; Obeid, et al., Science 259: 1769-1771,
1993), and the activation of nSMase has been shown to be critical
for TNF.alpha. induced apoptosis (Luberto, et al., J. Biol. Chem.
277: 41128-41139, 2002; Segui, et al., J. Clin. Invest. 108:
143-151, 2001). As a consequence, nSMase has also been proposed as
a target for drug discovery (Wascholowski and Giannis, Drug News
Perspect. 14: 581-90, 2001).
The sphingolipid signaling molecule, S1P, is produced from SPH
through the action of sphingosine kinase (SPHK). Two isoforms of
the kinase have been identified, SPHK1 and SPHK2 (Liu, J Biol Chem
275: 19513-20, 2000; Nava, et al., Exp Cell Res 281: 115-127,
2002). While CER and SPH are commonly associated with apoptosis,
conversely S1P is a mediator of cell proliferation and activation
of survival pathways (An, Ann N Y Acad Sci 905: 25-33, 2000;
Maceyka, et al., BBA 1585: 193-201, 2002; Zhang, et al., J. Cell
Biol. 114: 155-167, 1991). It has recently been appreciated as an
extracellular mediator that can activate a set of G Protein Coupled
Receptors (GPCRs) belonging to the S1P/LPA receptor family,
formerly known as Edg receptors (An, Ann N Y Acad Sci 905: 25-33,
2000; An, Goetzl, and Lee, J. cell biochem 30/31: 147-157, 1998;
Lee, et al., Science 279: 1552-1555, 1998; Okamoto, et al.,
Biochem. Biophys. Res. Commun. 260: 203-208, 1999); however,
intracellular actions of S1P have also been suggested (Van
Brocklyn, et al., J. Cell Biol. 142: 229-240, 1998). Moreover, it
has been suggested that the balance between CER/SPH levels versus
S1P provides a rheostat mechanism that decides whether a cell is
sent into the death pathway or is protected from apoptosis (Kwon,
et al., J Biol Chem 276: 10627-10633, 2001; Maceyka, et al., BBA
1585: 193-201, 2002; Pyne, Biochem J. 349: 385-402, 2000). The key
regulatory enzyme of the rheostat mechanism is SPHK whose role is
to convert the death-promoting sphingolipids (CER/SPH) in to the
growth-promoting S1P.
A landmark study first proposing SPHK as an oncogene was published
by a group from Adelaide demonstrating that NIH-3T3 fibroblasts
stably transfected with the kinase exhibited enhanced cell
proliferation accompanied by increased S1P production (Vadas and
Gamble, Circ. Res. 79: 1216-1217, 1996; Xia et al., Curr Biol 10:
1527-1530, 2000). In addition, the SPHK over-expressers escaped
contact inhibition, a property commonly exhibited by transformed
cells. This observation is consistent with a recent report showing
that S1P enhances metastatic potential of selected human cancer
cell lines (Igarashi, Ann. N.Y. Acad. Sci. 845: 19-31, 1998;
Takuwa, Biochim Biophys Acta. 1582: 112-120, 2002). Moreover, the
transfectants produced tumors when injected subcutaneous into
NOD/SCID mice. These results were recently confirmed in a study
showing that a small molecule inhibitor of SPHK given i.p. could
reduce tumor volume in SCID mice receiving subcutaneous injections
of JC mammary adenocarcinoma cells (French, et al., Cancer Res 63:
5962-5969, 2003). Significantly, the concept that SPHK could be a
novel oncogene was cemented by the finding that SPHK was
over-expressed in many solid tumors, such as those of the breast,
colon, lung, ovary, stomach, uterus, kidney, and rectum (French et
al. (2003), above). In addition, it has been demonstrated that
several human tumor-derived cell lines could be driven into
apoptosis when treated with the SPHK small molecule inhibitors, and
that their effectiveness could be accounted for by their abilities
to reduce S1P levels. Taken together, these findings demonstrate an
important concept that S1P is a growth factor likely produced by
tumor cells themselves and that lowering the concentration of S1P
may cause the apoptosis seen upon growth factor withdrawal.
S1P and Tumor Angiogenesis
Angiogenesis is the process by which new blood vessels are formed
from existing vasculature. Angiogenesis plays a critical role in
several physiological processes and is implicated in the
pathogenesis of a variety of disorders, including tumor growth,
invasion and metastasis. The angiogenesis process associated with
solid and circulating tumors (tumor angiogenesis) is considered to
be a crucial component of tumorigenesis and disease progression,
with the new blood vessels providing a growth advantage to tumor
cells compared to non-cancerous cells. Therefore, clinical control
of angiogenesis is a critical component for the treatment of cancer
and other angiogenesis-dependent diseases. Anti-angiogenic
therapeutics is particularly attractive because vascular
endothelial cells (ECs) do not mutate as easily as do cancer cells;
consequently, ECs are less likely than cancer cells to gain
resistance to prolonged therapy, making them good potential targets
for therapeutics.
Several growth factors have been implicated in cancerous
angiogenesis. The biolipid sphingosine-1-phosphate (S1P) was found
to be a mediator of many cellular processes important for cancer.
S1P exerts most of its effects as a specific ligand for a family of
G-protein-coupled receptors, designated S1P.sub.1-5. These
receptors regulate angiogenesis and vascular maturation, cell
movement, and lymphocyte trafficking. In contrast to S1P, the
precursors to S1P, sphingosine and ceramide, have been associated
with growth arrest and apoptosis. Finally, there is a complex
cross-talk between S1P and other pro-angiogenic growth factors such
as VEGF, EGF, PDGF, bFGF and IL-8. S1P, by binding to receptor
S1P.sub.1, transactivates growth factor receptor tyrosine kinase,
such as that found on VEGFR, EGFR, and PDGFR. The importance of S1P
in the angiogenesis-dependent tumors makes S1P an exceptional
target for cancer treatment. Based on these observations, an
antibody approach to neutralize the extracellular S1P could result
in a marked decrease in cancer progression in humans as a result of
inhibition of blood vessel formation with concomitant loss of the
nutrients and oxygen needed to support tumor growth. Furthermore,
recent research suggests that many angiogenesis inhibitors may also
act as anti-invasive and anti-metastatic compounds which could also
aid in the mitigation of the spread of cancer to sites distant from
the initial tumor.
A growing body of recent evidence implicating S1P as one of the
most potent pro-angiogenic agents comes from studies directly
comparing S1P with agents such as VEGF and bFGF. S1P stimulates DNA
synthesis and chemotactic motility of human venous endothelial
cells (HUVECs), while inducing differentiation of multicellular
structures, all of which is suggestive of S1P's role in early
blood-vessel formation (Argraves, et al., 2004; Lee et al., 1999;
Liu, et al., 2000). Also, S1P promotes the migration of bone
marrow-derived EC precursors to neovascularization sites (Annabi,
et al., 2003). Cells that over-express S1P, are resistant to the
anti-angiogenic agents thalidomide and Neovastat (Annabi et al.,
2003). In addition, it has been demonstrated that substantial
cross-talk exists between S1P and other pro-angiogenic growth
factors such as VEGF, EGF, PDGF, bFGF and IL-8. For example, S1P
transactivates EGF (Shida, et al., 2004) and VEGF2 receptors
(Spiegel & Milstien, 2003), and VEGF up-regulates S1P.sub.1
receptor expression (Igarashi, et al., 2003). Also, S1P, acting via
S1P.sub.1 and the "VEGF axis," is required for hind-limb
angiogenesis and neovascularization (Chae, et al., 2004).
The anti-angiogenic approach to cancer has been greatly advanced by
the recent FDA approval of the anti-angiogenic drug, bevacizumab
(Avastin.RTM., Genentech) to treat colon cancer as an adjunct to
cytotoxic chemotherapy.
An anti-S1P murine MAb, LT1002 was developed recently with high
binding affinity and specificity to S1P. LT1002 was shown to
significantly slow tumor progression and associated angiogenesis in
several animal models of human cancer. In addition, LT1002
attenuated choroidal neovascularization (CNV) lesion formation in
the well-established model of angiogenesis for age-related macular
degeneration (AMD). CNV occurs in diseases in which there are
abnormalities of Bruch's membrane and the retinal pigmented
epithelium. The most common disease of this type is AMD, the most
prevalent cause of severe loss of vision in elderly patients. These
results suggested that LT1002 has several mechanisms of action,
including: (1) direct effects on tumor cell growth, (2) an indirect
anti-angiogenic effect on vascular endothelia cells, and (3) an
indirect anti-angiogenic effect of preventing the release and
action of other pro-angiogenic growth factors.
The most direct in vivo evidence that S1P contributes to tumor
angiogenesis comes from our recent publication that focused on a
murine monoclonal antibody (mAb) designed to neutralize
extracellular S1P by molecular absorption (Visentin, et al., 2006).
In various in vitro assays using HUVECs, the anti-S1P mAb
neutralized tube formation, migration of vascular endothelial cells
and protection from cell death, each of which is S1P-induced. S1P
increased new capillary growth into Matrigel plugs implanted in
mice, an effect that was neutralized by the systemic administration
of the anti-S1P mAb. The mAb substantially neutralized bFGF- and
VEGF-induced angiogenesis in a murine Matrigel plug assay, and the
antibody mitigated S1P stimulated the release of pro-angiogenic
cytokines (VEGF, IL-8, IL-6) from tumor cells in vitro and in vivo.
Importantly, mice xenografted with orthotopically-placed human
cancer cells exhibited substantial retardation of tumor progression
with anti-S1P mAb treatment. This was demonstrated in murine models
of human breast, ovarian and lung cancer and in one allograft model
of murine melanoma (Visentin, et al., 2006).
The use of monoclonal antibodies (mAbs) as a therapeutic treatment
for a variety of diseases and disorders is rapidly increasing
because they have been shown to be safe and efficacious therapeutic
agents. Approved therapeutic mAbs include Avastin.RTM.,
Erbitux.RTM., and Rituxan.RTM.. Additional mAbs are in various
phases of clinical development for a variety of diseases with the
majority targeting various forms of cancer. In general, monoclonal
antibodies are generated in non-human mammals. The therapeutic
utility of murine monoclonal antibodies is limited, however,
principally due to the fact that human patients mount their own
antibody response to murine antibodies. This response, the
so-called HAMA (human anti-mouse antibody) response, results in the
eventual neutralization and rapid elimination of murine mAbs. This
limitation has been overcome with the development of a process
called "humanization" of murine antibodies. Humanization greatly
lessens the development of an immune response against the
administered therapeutic MAb and thereby avoids the reduction of
half-life and therapeutic efficacy consequent on HAMA. For the most
part, the humanization process consists of grafting the murine
complementary determining regions (CDRs) into the framework region
(FR) of a human immunoglobulin. This strategy is referred to as
"CDR grafting". "Backmutation" to murine amino acid residues of
selected residues in the human FR is often required to regain
affinity that is lost in the initial grafted construct.
The manufacture of mAbs is a complex process that stems from the
variability of the protein itself. The variability of mAbs can be
localized to the protein backbone and/or to the carbohydrate
moiety. The heterogeneity can be attributed to the formation of
alternative disulfide pairings, deamidation and the formation of
isoaspartyl residues, methionine and cysteine oxidation,
cyclization of N-terminal glutamine residues to pyroglutamate and
partial enzymatic cleavage of C-terminal lysines by mammalian
carboxypeptidases. Engineering is commonly applied to antibody
molecules to improve their properties, such as enhanced stability,
resistance to proteases, aggregation behavior and enhance the
expression level in heterologous systems.
Here, the humanization of the murine MAb against S1P is described.
The overall strategy consisted of grafting the six CDRs from LT1002
into a human framework. Further modifications were engineered to
further refine and optimize the antibody performance. The humanized
MAb presented the same characteristics as the LT1002 and is thus
suitable for testing in clinical trials.
S1P and Fibrosis
Fibroblasts, particularly myofibroblasts, are key cellular elements
in scar formation in response to cellular injury and inflammation
[Tomasek, et al. (2002), Nat Rev Mol Cell Biol, vol 3: 349-63, and
Virag and Murry (2003), Am J Pathol, vol 163: 2433-40]. Collagen
gene expression by myofibroblasts is a hallmark of remodeling and
necessary for scar formation [Sun and Weber (2000), Cardiovasc Res,
vol 46: 250-6, and Sun and Weber (1996), J Mol Cell Cardiol, vol
28: 851-8]. S1P promotes wound healing by activating fibroblast
migration and proliferation while increasing collagen production
[Sun, et al. (1994), J Biol Chem, vol 269: 16512-7]. S1P produced
locally by damaged cells could be responsible for the maladaptive
wound healing associated with remodeling and scar formation. Thus
it is believed that S1P inhibitors are useful in diseases or
conditions characterized, at least in part, by aberrant
fibrogenesis or fibrosis. Herein, "fibrogenesis" is defined as
excessive activity or number of fibroblasts, and "fibrosis" is
defined as excessive activity or number of fibroblasts that leads
to excessive or inappropriate collagen production and scarring,
destruction of the physiological tissue structure and/or
inappropriate contraction of the matrix leading to such pathologies
as retinal detachment or other processes leading to impairment of
organ function.
S1P and fibroblast collagen expression: S1P promotes the
differentiation of quiescent fibroblasts to active myofibroblasts
which exhibit enhanced collagen expression during scar formation
[Urata, et al. (2005), Kobe J Med Sci, vol 51: 17-27]. Concurrent
with the proliferation and migration of fibroblasts into the
scarring zone, myofibroblasts deposit a temporary granular network
consisting primarily of osteopontin and fibronectin [Sun and Weber
(2000), Cardiovasc Res, vol 46: 250-6]. As remodeling proceeds, the
temporary matrix is absorbed and a collagen network established
[Sun and Weber (2000), Cardiovasc Res, vol 46: 250-6]. We have
demonstrated that S1P promotes collagen production by
myofibroblasts. TGF.beta., a well-known fibrotic mediator, has been
shown to up-regulate several pro-fibrotic proteins, convert
fibroblasts to myofibroblasts and stimulate inflammatory protein
expression possibly through the action of S1P [Squires, et al.
(2005), J Mol Cell Cardiol, vol 39: 699-707 and Butt, Laurent and
Bishop (1995), Eur J Cell Biol, vol 68: 330-5]. Up-regulation of
TIMP1, a signaling molecule implicated in TGF.beta.-stimulated
differentiation of fibroblasts to myofibroblasts, is blocked by
siRNA against SPHK1 [Yamanaka, et al., J Biol. Chem. 2004 Dec. 24;
279(52):53994-4001], suggesting that a humanized version of the
anti-S1P antibody could mitigate the profibrotic effects of
TGF.beta. as well as mitigating the fibrogenic effects of S1P
itself.
Minimizing maladaptive scarring is believed to be useful in
treatment of fibrotic diseases and conditions, including but not
limited to ocular and cardiovascular diseases, wound healing, and
scleroderma.
Anti-S1P Antibodies for the Treatment of Scleroderma
The compositions and methods of the invention will be useful in
treating disorders and diseases characterized, at least in part, by
aberrant neovascularization, angiogenesis, fibrogenesis, fibrosis,
scarring, inflammation, and immune response. One such disease is
scleroderma, which is also referred to as systemic sclerosis.
Scleroderma is an autoimmune disease that causes scarring or
thickening of the skin, and sometimes involves other areas of the
body, including the lungs, heart, and/or kidneys. Scleroderma is
characterized by the formation of scar tissue (fibrosis) in the
skin and organs of the body, which can lead to thickening and
firmness of involved areas, with consequent reduction in function.
Today, about 300,000 Americans have scleroderma, according to the
Scleroderma Foundation. One-third or less of those affected have
widespread disease, while the remaining two-thirds primarily have
skin symptoms. When the disease affects the lungs and causing
scarring, breathing can become restricted because the lungs can no
longer expand as they should. To measure breathing capability,
doctors use a device that assesses forced vital capacity (FVC). In
people with an FVC of less than 50 percent of the expected reading,
the 10-year mortality rate from scleroderma-related lung disease is
about 42 percent. One reason the mortality rate is so high is that
no effective treatment is currently available.
As described in the examples of this application, existing evidence
indicates that S1P is a pro-fibrotic growth factor that can
contribute to fibroblast activation, proliferation, and the
resulting increased fibroblast activity associated with maladaptive
scarring and remodeling. Moreover, potential roles for S1P in
activity of skin and other types of fibroblasts have been
demonstrated. For example, it has been shown that bioactive lipids
stimulate the migration of murine skin fibroblasts (Hama, et al., J
Biol Chem. 2004 Apr. 23; 279(17):17634-9), and human skin
fibroblasts express several S1P receptor subtypes (Zhang, et al.,
Blood. 1999 May 1; 93(9):2984-90). In addition to the many direct
effects of S1P on fibroblast activity, S1P also may have many
potential indirect effects on fibroblast activity. For example, S1P
may facilitate the action of other well-known pro-fibrotic factors,
such as TGF-.beta. and platelet derived growth factor (PDGF).
TGF-.beta. is one of the most widely studied and recognized
contributors to fibrosis (Desmouliere, et al., J Cell Biol 122:
103-111, 1993). TGF-.beta. upregulates SphK1 expression and
activity leading to increased expression of tissue inhibitors of
metalloproteinases 1 (TIMP-1), a protein that inhibits ECM
degradation (Yamanaka, et al., J Biol Chem 279: 53994-54001, 2004).
Increased expression of TIMP-1 is linked to interstitial fibrosis
and diastolic dysfunction in heart failure patients (Heymans, et
al., Am J Pathol 166: 15-25, 2005). Conversely, S1P stimulates
expression and release of TGF-.beta. (Norata, et al., Circulation
111: 2805-2811, 2005). There is also distinct evidence of crosstalk
between S1P and PDGF. S1P directly stimulates expression of PDGF
(Usui, et al., J Biol Chem 279: 12300-12311, 2004). In addition,
the S1P.sub.1 receptor and the PDGF receptor bind one another and
their association is necessary for PDGF activation of downstream
signaling which contributes to proliferation and migration of
various cell types (Long, et al., Prostaglandins Other Lipid Mediat
80: 74-80, 2006; Baudhuin et al., Faseb J 18: 341-343, 2004). As
such, the effects of TGF-.beta. and PDGF on fibrosis may be due in
part to crosstalk with the S1P signaling pathway. As such, the
compositions and methods of the invention can be used to treat
scleroderma, particularly by decreasing the effective in vivo
concentration of a particular target lipid, for example, S1P.
Systemic scleroderma is thought to be exacerbated by stimulatory
autoantibodies against PDGF receptors (Baroni, et al., N Engl J
Med. 2006 v354(25):2667-76), and PDGF receptors are up-regulated in
scleroderma fibroblasts in response to TGF-.beta. (Yamakage, et
al., J Exp Med. 1992 May 1; 175(5):1227-34). Because of the
substantial cross-talk among the S1P, PDGF and TGF-.beta. signaling
systems, blocking S1P bioactivity with and anti-S1P agent (e.g., an
anti-S1P mAb) could indirectly mitigate the pro-sclerotic effects
of PDGF and TGF-.beta.. Moreover, treatment with such an anti-S1P
agent could benefit scleroderma patients by mitigating the direct
effects of S1P on skin and other forms of fibroblasts that
contribute to disease progression.
S1P and Ocular Diseases and Conditions
Pathologic or aberrant angiogenesis/neovascularization, aberrant
remodeling, fibrosis and scarring and inflammation occur in
association with retinal and ocular diseases such as age-related
macular degeneration (AMD), diabetic retinopathy (DR), and in
retinopathy of prematurity (ROP) and other developmental disorders
[Eichler, et al. (2006), Curr Pharm Des, vol 12: 2645-60], as well
as being a result of infections and mechanical injury to the eye
[Ciulla, et al. (2001), Curr Opin Opthalmol, vol 12: 442-9 and Dart
et al (2003), Eye, vol 17: 886-92]. It is believed that antibodies
against S1P will be useful in treating ocular diseases for which
pathologic or aberrant angiogenesis/neovascularization, aberrant
remodeling, fibrosis, and scarring or inflammation are a
component.
Angiogenesis/Neovascularization of the Eye:
Pathologic ocular angiogenesis is a leading cause of blindness in a
variety of clinical conditions. Choroidal neovascularization (CNV)
occurs in a number of ocular diseases, the most prevalent of which
is the exudative or "wet" form of AMD. As a result of an
increasingly aged population, AMD is a modern day epidemic and the
leading cause of blindness in the western world in patients over
age 60. Despite the epidemic of vision loss caused by AMD, only a
few therapies, mostly anti-VEGF based, can slow the progression of
AMD and even fewer can reverse vision loss [Bylsma and Guymer
(2005), Clin Exp Optom., vol 88: 322-34, Gryziewicz (2005), Adv
Drug Deliv Rev, vol 57: 2092-8, and Liu and Regillo (2004), Curr
Opin Opthalmol, vol 15: 221-6.]. Therefore, discovering new
treatments for pathologic neovascularization is extremely
important.
AMD is used here solely for illustrative purposes in describing
ocular conditions relating to aberrant
angiogenesis/neovascularization, aberrant remodeling, fibrosis and
scarring, and inflammation, which conditions are found in other
ocular diseases and disorders as disclosed and claimed herein. AMD
involves age-related pathologic changes [Tezel, Bora, and Kaplan
(2004), Trends Mol Med, vol 10: 417-20 and Zarbin (2004), Arch
Opthalmol, 122: 598-614]. Multiple theories exist but, the exact
etiology and pathogenesis of AMD are still not well understood.
Aging is associated with cumulative oxidative injury, thickening of
Bruch's membrane and drusen formation. Oxidative stress results in
injury to retinal pigment epithelial (RPE) cells and, in some
cases, the choriocapillaris [Zarbin (2004), Arch Opthalmol, vol
122: 598-614, and Gorin, et al. (1999), Mol Vis., vol 5: 29].
Injury to RPE likely elicits a chronic inflammatory response within
Bruchs membrane and the choroid [Johnson et al. (2000), Exp Eye
Res., vol 70: 441-9]. This injury and inflammation fosters and
potentates retinal damage by stimulating CNV and atrophy [Zarbin
(2004), Arch Opthalmol, vol 122: 598-614, and Witmer, et al.
(2003), Prog Retin Eye Res, vol 22: 1-29]. CNV results in defective
and leaky blood vessels (BV) that are likely to be recognized as a
wound [Kent and Sheridan (2003), Mol Vis, vol 9: 747-55]. Wound
healing arises from the choroid and invades the subretinal space
through Bruchs membrane and the RPE. Wound healing responses are
characterized by a typical early inflammation response, a prominent
angiogenic response and tissue formation followed by end-stage
maturation of all involved elements. Wound remodeling may
irreversibly compromise photoreceptors and RPEs thereby, justifying
the need to treat CNV with more than anti-angiogenic therapies [La
Cour, Kiilgaard, and Nissen (2002), Drugs Aging, vol 19:
101-33.12].
Alterations in the normal retinal and sub-retinal architecture as a
result of CNV related fibrosis, edema and inflammation individually
or cumulatively, leads to AMD related visual loss [Tezel and Kaplan
(2004), Trends Mol Med, vol 10: 417-20, and Ambati, et al. (2003),
Surv Opthalmol, vol 48: 257-93]. The multiple cellular and cytokine
interactions which are associated with exudative AMD greatly
complicate the search for effective treatments. While CNV and edema
are manageable in part by anti-VEGF therapeutics, potential
treatments to mitigate scar formation and inflammation have not
been adequately addressed [Bylsma and Guymer (2005), Clin Exp
Optom, vol 88: 322-34, and Pauleikhoff (2005), Retina, vol 25:
1065-84]. As long as the neovascular complex remains intact, as
appears to be the case in patients treated with anti-VEGF agents,
the potential for subretinal fibrosis and future vision loss
persists.
Anti-VEGF-A therapies represent a recent, significant advance in
the treatment of exudative AMD. However, the phase III VISION Trial
with PEGAPTANIB, a high affinity aptamer which selectively inhibits
the 165 isoform of VEGF-A, demonstrated that the average patient
continues to lose vision and only a small percent gained vision
[Gragoudas, et al. (2004), N Engl J Med, vol 351: 2805-16].
Inhibition of all isoforms of VEGF-A (pan-VEGF inhibition) with the
antibody fragment RANIBIZUMAB yielded much more impressive results
[Brown, et al., N Eng Med (2006), vol. 355:1432-44, Rosenfeld, et
al. N Eng J Med (2006), vol. 355:1419-31]. The 2 year MARINA trial
and the I year ANCHOR trial demonstrated that approximately 40% of
patients achieve some visual gain. Although these results represent
a major advance in our ability to treat exudative AMD, they also
demonstrate that 60% of patients do not have visual improvement.
Furthermore, these patients had to meet strictly defined inclusion
and exclusion criteria. The results in a larger patient population
may be less robust.
There is still a well-defined need to develop further therapeutic
agents that target other steps in the development of CNV and the
processes that ultimately lead to photoreceptor destruction. First,
the growth of choroidal BVs involves an orchestrated interaction
among many mediators, not just VEGF, offering an opportunity to
modulate or inhibit the entire process [Gragoudas, et al. (2004), N
Engl J Med, vol 351: 2805-16]. Second, exudative AMD is comprised
of vascular and extravascular components. The vascular component
involves vascular endothelial cells (EC), EC precursors and
pericytes. The extravascular component, which volumetrically
appears to be the largest component, is composed of inflammatory,
glial, and retinal pigment epithelium (RPE) cells and fibroblasts.
Tissue damage can result from either component. These other aspects
of the pathologic process are not addressed by current anti-VEGF
treatments. Targeting additional elements of the angiogenic cascade
associated with AMD could provide a more effective and synergistic
approach to therapy [Spaide, R F (2006), Am J Opthalmol, vol 141:
149-156].
Inflammation in Ocular Disease:
There is increasing evidence that inflammation, specifically
macrophages and the complement system [Klein, et al. (2005),
Science, vol 308: 385-9; and Hageman, et al. (2005), Proc Natl Acad
Sci USA, vol 102: 7227-32], plays an important role in the
pathogenesis of exudative AMD. Histopathology of surgically excised
choroidal neovascular membranes demonstrates that macrophages are
almost universally present [Grossniklaus, et al. (1994),
Ophthalmology, vol 101: 1099-111, and Grossniklaus, et al. (2002),
Mol Vis, vol 8: 119-26]. There is mounting evidence that
macrophages may play an active role in mediating CNV formation and
propagation [Grossniklaus, et al. (2003), Mol Vis, vol 8: 119-26;
Espinosa-Heidmann, et al. (2003), Invest Opthalmol Vis Sci, vol 44:
3586-92; Oh, et al. (1999), Invest Opthalmol Vis Sci, vol 40:
1891-8; Cousins, et al. (2004), Arch Opthalmol, vol 122: 1013-8;
Forrester (2003), Nat Med, vol 9: 1350-1, and Tsutsumi, et al.
(2003), J Leukoc Biol, vol 74: 25-32] by multiple effects which
include secretion of enzymes that can damage cells and degrade
Bruchs membrane as well as release pro-angiogenic cytokines [Otani,
et al. (1999), Opthalmol Vis Sci, vol 40: 1912-20, and Amin,
Puklin, and Frank (1994), Invest Opthalmol Vis Sci, vol 35:
3178-88]. At the site of injury, macrophages exhibit
micro-morphological signs of activation, such as degranulation [Oh,
et al. (1999), Invest Opthalmol Vis Sci, vol 40: 1891-8, and
Trautmann et al. (2000), J Pathol, vol 190: 100-6]. Thus it is
believed that a molecule which limited macrophage infiltration into
to the choroidal neovascular complex may help limit CNV
formation.
Choroidal Neovascularization and Blood Vessel Maturation in Ocular
Disease:
Angiogenesis is an essential component of normal wound healing as
it delivers oxygen and nutrients to inflammatory cells and assists
in debris removal [Lingen (2001), Arch Pathol Lab Med, vol 125:
67-71]. Progressive angiogenesis is composed of two distinct
processes: Stage I: Migration of vascular ECs, in response to
nearby stimuli, to the tips of the capillaries where they
proliferate and form luminal structures; and Stage II: Pruning of
the vessel network and optimization of the vasculature [Guo, et al.
(2003), Am J Pathol, vol 162: 1083-93].
Stage I: Neovascularization. Angiogenesis most often aids wound
healing. However, new vessels, when uncontrolled, are commonly
defective and promote leakage, hemorrhaging, and inflammation.
Diminishing dysfunctional and leaky BVs, by targeting
pro-angiogenic GFs, has demonstrated some ability to slow the
progression of AMD [Pauleikhoff (2005), Retina, vol 25: 1065-84.14,
and van Wijngaarden, Coster, and Williams (2005), JAMA, vol 293:
1509-13].
Stage II: Blood vessel maturation and drug desensitization.
Pan-VEGF inhibition appears to exert its beneficial effect mostly
via an anti-permeability action resulting in resolution of intra-
and sub-retinal edema, as the actual CNV lesion does not markedly
involute. The lack of marked CNV involution may in part be a result
of maturation of the newly formed vessels due to pericyte coverage.
Pericytes play a critical role in the development and maintenance
of vascular tissue. The presence of pericytes seems to confer a
resistance to anti-VEGF agents and compromise their ability to
inhibit angiogenesis [Bergers and Song (2005), Neuro-oncol, vol 7:
452-64; Yamagishi and Imaizumi (2005), Int J Tissue React, vol 27:
125-35; Armulik, Abramsson and Betsholtz (2005), Circ Res, vol 97:
512-23; Ishibashi et al. (1995), Arch Opthalmol, vol 113: 227-31].
An agent that has an inhibitory effect on pericyte recruitment
would likely disrupt vascular channel assembly and the maturation
of the choroidal neovascular channels thereby perpetuating their
sensitivity to anti-angiogenic agents.
Remodeling of the vascular network involves adjustments in blood
vessel (BV) density to meet nutritional needs [Gariano and Gardner
(2005), Nature, 438: 960-6]. Periods of BV immaturity corresponds
to a period in which new vessels are functioning but have not yet
acquired a pericyte coating [Benjamin, Hemo, and Keshet (1998),
Development, 125: 1591-8, and Gerhardt and Betsholtz (2003), Cell
Tissue Res, 2003. 314: 15-23]. This delay is essential in providing
a window of plasticity for the fine tuning of the developing
vasculature according to the nutritional needs of the retina or
choroid.
The bioactive lipid sphingosine-1-phosphate (S1P), VEGF, PDGF,
angiopoietins (Ang) and other growth factors (GF) augment blood
vessel growth and recruit smooth muscle cells (SMC) and pericytes
to naive vessels which promote the remodeling of emerging vessels
[Allende and Proia (2002), Biochim Biophys Acta, vol 582: 222-7;
Gariano and Gardner (2005), Nature, vol 438: 960-6; Grosskreutz, et
al. (1999), Microvasc Res, vol 58: 128-36; Nishishita, and Lin
(2004), J Cell Biochem, vol 91: 584-93, and Erber, et al. (2004),
FASEB J, vol 18: 338-40.32]. Pericytes, most likely generated by in
situ differentiation of mesenchymal precursors at the time of EC
sprouting or from the migration and de-differentiation of arterial
smooth muscle cells, intimately associate and ensheath ECs
resulting in overall vascular maturity and survival [Benjamin,
Hemo, and Keshet (1998), Development, vol 125: 1591-8]. Recent
studies have demonstrated that S1P, and the S1P1 receptor, are
involved in cell-surface trafficking and activation of the
cell-cell adhesion molecule N-cadherin [Paik, et al. (2004), Genes
Dev, vol 18: 2392-403]. N-cadherin is essential for interactions
between EC, pericytes and mural cells which promote the development
of a stable vascular bed [Gerhardt and Betsholtz (2003), Cell
Tissue Res, vol 314: 15-23]. Global deletion of the S1P1 gene
results in aberrant mural cell ensheathment of nascent BVs required
for BV stabilization during embryonic development [Allende and
Proia (2002), Biochim Biophys Acta, vol 1582: 222-7]. Local
injection of siRNA to S1PI suppresses vascular stabilization in
tumor xenograft models [Chae, et al. (2004), J Clin Invest, vol
114: 1082-9]. Transgenic mouse studies have demonstrated that VEGF
and PDGF-B promote the maturation and stabilization of new BVs
[Guo, et al. (2003), Am J Pathol, 162: 1083-93, and Gariano and
Gardner (2005), Nature, vol 438: 960-6.50]. VEGF up-regulates Ang-1
(mRNA and protein) [Asahara, et al. (1998), Circ Res, vol 83:
233-40]. Ang-1 plays a major role in recruiting and sustaining
peri-endothelial support by pericytes [Asahara, et al. (1998), Circ
Res, vol 83: 233-40]. Intraocular injection of VEGF accelerated
pericyte coverage of the EC plexus [Benjamin, Hemo, and Keshet
(1998), Development, vol 125: 1591-8]. PDGF-B deficient mouse
embryos lack micro-vascular pericytes, which leads to edema,
micro-aneurisms and lethal hemorrhages [Lindahl, et al. (1997),
Science, vol 277: 242-5]. Murine pre-natal studies have
demonstrated that additional signals are required for complete
VEGF- and PDGF-stimulation of vascular bed maturation. Based upon
the trans-activation of S1P noted above, this factor could be S1P
[Erber et al. (2004), FASEB J, vol 18: 338-40]. Vessel
stabilization and maturation is associated with a loss of
plasticity and the absence of regression to VEGF and other GF
withdrawal and resistance to anti-angiogenic therapies [Erber, et
al. (2004), FASEB J, vol 18: 338-40, and Hughes and Chan-Ling
(2004), Invest Opthalmol Vis Sci, vol 45: 2795-806]. Resistance of
BVs to angiogenic inhibitors is conferred by pericytes that
initially stabilize matured vessels and those that are recruited to
immature vessels upon therapy [Erber, et al. (2004), FASEB J, vol
18: 338-40]. After ensheathment of the immature ECs, the pericytes
express compensatory survival factors (Ang-1 and PDGF-B) that
protect ECs from pro-apoptotic agents.
Edema and Vascular Permeability in Ocular Disease:
CNV membranes are composed of fenestrated vascular ECs that tend to
leak their intravascular contents into the surrounding space
resulting in subretinal hemorrhage, exudates and fluid accumulation
[Gerhardt and Betsholtz (2003), Cell Tissue Res, vol 14: 15-23].
For many years the CNV tissue itself, and more recently
intra-retinal neovascularization, have been implicated as being
responsible for the decrease in visual acuity associated with AMD.
It is now thought however, that macular edema caused by an increase
in vascular permeability (VP) and subsequent breakdown of the blood
retinal barrier (BRB), plays a major role in vision loss associated
with AMD and other ocular diseases, including blindness associated
with diabetes. [Hughes and Chan-Ling (2004), Invest Opthalmol V is
Sci, vol 45: 2795-806; Felinski and Antonetti (2005), Curr Eye Res,
vol 30: 949-57; Joussen, et al. (2003), FASEB J, vol 17: 76-8, and
Strom, et al. (2005), Invest Opthalmol Vis Sci, vol 46: 3855-8]. In
particular, diabetic retinopathy (DR) and diabetic macular edema
(DME) are common microvascular complications in patients with
diabetes and are the most common causes of diabetes-associated
blindness. DME results from increased microvascular permeability.
Joussen, et al. (2003), FASEB J, vol 17: 76-8. Together these are
the most common cause of new blindness in the working-age
population. It is believed that compounds, such as antibodies that
target S1P, will be therapeutically useful for these
conditions.
Examples of several classes of ocular diseases that may be treated
in accordance with the invention are described below. It will be
appreciated that many disease and conditions are characterized, at
least in part, by multiple pathological processes (for example,
both pathological neovascularization and scarring) and that the
classifications provided herein are for descriptive convenience and
do not limit the invention.
a. Ischemic Retinopathies Associated with Pathologic
Neovascularization and Diseases Characterized by Epiretinal and or
Subretinal Membrane Formation.
Ischemic retinopathies (IR) are a diverse group of disorders
characterized by a compromised retinal blood flow. Examples of IR
include diabetic retinopathy (DR), retinopathy of prematurity
(ROP), sickle cell retinopathy and retinal venous occlusive
disease. All of these disorders can be associated with a VEGF
driven proliferation of pathological retinal neovascularization
which can ultimately lead to intraocular hemorrhaging, epi-retinal
membrane formation and tractional retinal detachment. Idiopathic
epi-retinal membranes (ERMs), also called macular pucker or
cellophane retinopathy, can cause a reduction in vision secondary
to distortion of the retinal architecture. These membranes
sometimes recur despite surgical removal and are sometimes
associated with retinal ischemia. VEGF and its receptors are
localized to ERMs. The presence of VEGF in membranes associated
with proliferative diabetic retinopathy, proliferative
vitreoretinopathy, and macular pucker further suggests that this
cytokine plays an important role in angiogenesis in ischemic
retinal disease and in membrane growth in proliferative
vitreoretinal disorders. In addition, VEGF receptors VEGFR1 and
VEGFR2 are also identified on cells in ERMs. These data show that
VEGF may be an autocrine and/or paracrine stimulator that may
contribute to the progression of vascular and avascular ERMs. PDGF
and its receptors [Robbins, et al. (1994), Invest Opthalmol Vis
Sci; vol 35: 3649-3663] has been described in eyes with
proliferative retinal diseases [Cassidy, et al. (1998), Br J
Ophthamol; vol 82: 181-85, and Freyberger, et al. (2000), Exp Clin
Endocrinol Diabetes, vol 108: 106-109]. These findings suggest that
PDGF ligands and receptors are widespread in proliferative retinal
membranes of different origin and suggest that autocrine and
paracrine stimulation with PDGF may be involved in ERM
pathogenesis. Transforming growth factor-p (TGF-.beta.) is involved
in the formation of ERMs [Pournaras, et al. (1998), Klin Monatsbl
Augenheilkd, vol 212: 356-358] as demonstrated by TGF staining and
immunoreactivity. In addition, TGF-.beta. receptor II is expressed
in myofibroblasts of ERM of diabetic and PVR membranes. These
results suggest that TGF-.beta., produced in multiple cell types in
retina and ERMs, is an attractive target for the treatment of PVR,
diabetic and secondary ERMs. Interleukin-6 (IL-6) has been reported
to be increased in human vitreous in proliferative diabetic
retinopathy (PDR) [La Heij, et al. (2002), Am J Ophthal, 134:
367-375] and in one study 100% of the diabetic ERMs studied
expressed IL-6 protein [Yamamoto, et al. (2001) Am J Ophthal, vol
132: 369-377].
Exogenous administration of basic fibroblastic growth factor (bFGF)
has been shown to induce endothelial proliferation and VEGF
expression [Stavri, et al. (1995), Circulation, vol 92: 11-14].
Consistent with these observations, bFGF concentration is increased
in vitreous samples from patients with PDR [Sivalingam, et al.
(1990), Arch Opthalmol, vol 108: 869-872, and Boulton, et al.
(1997), Br J Opthalmol, vol 81: 228-233]. bFGF is also involved in
the formation of ERMs [Hueber, et al. (1996), Int. Opthalmol, vol
20: 345-350] demonstrated bFGF in 8 out of 10 PDR membranes
studied. Moreover, these workers found positive staining for the
corresponding receptor, FGFR1. Immunoreactivity for bFGF has also
been demonstrated in nonvascular idiopathic ERMs. These results
implicate bFGF in the formation of both vascular and avascular
ERMs. Harada, et al. (2006), Prog in Retinal and Eye Res, vol 25;
149-164. Elevated bFGF has also been detected in the serum of
patients with ROP (Becerril, et al. (2005), Opthalmology, vol 112,
2238].
Given the known pleotropic effects of S1P and its interactions with
VEGF, bFGF, PDGF, TGF-.beta. and IL-6, it is believed that an agent
that binds, antagonizes, inhibits the effects or the production of
S1P will be effective at suppressing pathologic retinal
neovascularization in ischemic retinopathies and posterior segment
diseases characterized by vascular or avascular ERM formation.
Other ocular conditions characterized, at least in part, by
aberrant neovascularization or angiogenesis include age-related
macular degeneration, corneal graft rejection, neovascular
glaucoma, contact lens overwear, infections of the cornea,
including herpes simplex, herpes zoster and protozoan infection,
pterygium, infectious uveitis, chronic retinal detachment, laser
injury, sickle cell retinopathy, venous occlusive disease,
choroidal neovascularization, retinal angiomatous proliferation,
and idiopathic polypoidal choroidal vasculopathy.
b. Proliferative Vitreoretinopathy (PVR).
PVR is observed after spontaneous rhegmatogenous retinal detachment
and after traumatic retinal detachment. It is a major cause of
failed retinal detachment surgery. It is characterized by the
growth and contraction of cellular membranes on both sides of the
retina, on the posterior vitreous surface and the vitreous base.
This excessive scar tissue development in the eye may lead to the
development of tractional retinal detachment, and therefore
treatments directed at the prevention or inhibition of
proliferative vitreoretinopathy (PVR) are a logical principle of
management of retinal detachment. Histopathologically PVR is
characterized by excessive collagen production, contraction and
cellular proliferation [Michels, Retinal Detachment 2nd Edition.
Wilkinsin C P, Rice T A Eds, Complicated types of retinal
detachment, pp 641-771, Mosby St Louis 1997]. Cellular types
identified in PVR membranes include mainly retinal pigmented
epithelial cells, fibroblasts, macrophages and vascular endothelial
cells [Jerdan, et al. (1989), Opthalmology, vol 96: 801-10, and
Vidinova, et al. (2005), Klin Monatsbl Augenheilkd; vol
222:568-571]. The pathophysiology of this excessive scarring
reaction appears to be mediated by a number of cytokines including
platelet derived growth factor (PDGF), transforming growth factor
(TGF) beta, basic fibroblastic growth factor (bFGF), interleukin-6
(IL)-6, and interleukin-8 (IL)-8 [Nagineni, et al. (2005), J Cell
Physiol, vol 203: 35-43; La Heij, et al (2002), Am J Opthalmol,
134: 367-75; Planck, et al. (1992), Curr Eye Res; vol 11: 1031-9;
Canataroglu et al. (2005) Ocul Immunol Inflamm; vol 13: 375-81, and
Andrews, et al. (1999), Opthalmol Vis Sci; vol 40: 2683-9].
Inhibition of these cytokines may help prevent the development of
PVR if given in a timely fashion or limit its severity [Akiyama, et
al (2006), J Cell Physiol, vol 207:407-12, and Zheng, et al (2003),
Jpn J Opthalmolm, vol 47:158-65].
Sphingosine-1-Phosphate (S1P) is a bioactive lysolipid with
pleotrophic effects. It is pro-angiogenic, pro inflammatory
(stimulates the recruitment of macrophages and mast cells) and
pro-fibrotic (stimulates scar formation). S1P generally stimulates
cells to proliferate and migrate and is anti-apoptotic. S1P
achieves these biologically diverse functions through its
interactions with numerous cytokines and growth factors. Inhibition
of S1P via a monoclonal antibody (SPHINGOMAB) has been demonstrated
to block the functions of vascular endothelial growth factor
(VEGF), bFGF, IL-6, and IL-8 [Visentin, B et al. (2006), Cancer
Cell, vol 9: 1-14]. Binding of S1P to the S1P.sub.1 receptor can
also increase PDGF production; therefore an agent that binds S1P
would also be expected to diminish PDGF production [Milstien and
Spiegel (2006), Cancer Cell, vol 9:148-150]. As shown in the
Examples below, it has now been demonstrated that in vitro S1P
transforms human RPE cells into a myofibroblast-like phenotype
similar to the type seen in PVR. Given the pathophysiology that
ultimately results in the excessive scarring seen in PVR and the
known effects of S1P on these same key mediators, it is believed
that an agent that binds, antagonizes, or inhibits the effects or
the production of S1P will be effective at eliminating or
minimizing the development of PVR and its severely damaging effects
on the eye.
c. Uveitis.
Uveitis is an inflammatory disorder of the uveal tract of the eye.
It can affect the front (anterior) or back (posterior) of the eye
or both. It can be idiopathic or infectious in etiology and can be
vision-threatening. Idiopathic uveitis has been associated with
increased CD4+ expression in the anterior chamber [Calder, et al.
(1999), Invest Opthalmol Vis Sci, vol 40: 2019-24]. Data also
suggests a pathologic role of the T lymphocyte and its
chemoattractant IP-10 in the pathogenesis of uveitis [Abu El-Asrar
(2004), Am J Opthalmol, vol 138: 401-11]. Other chemokines in acute
anterior uveitis include macrophage inflammatory proteins, monocyte
chemoattractant protein-1 and IL-8. These cytokines probably play a
critical role in leukocyte recruitment in acute anterior uveitis.
Verma, et al. (1997), Curr Eye Res; vol 16; 1202-8. Given the
profound and pleiotropic effects of the S1P signaling cascade, it
is believed that SPHINGOMAB and other immune moieties that reduce
the effective concentration of bioactive lipid would serve as an
effective method of reducing or modulating the intraocular
inflammation associated with uveitis.
d. Refractive Surgery.
The corneal wound healing response is of particular relevance for
refractive surgical procedures since it is a major determinant of
safety and efficacy. These procedures are performed for the
treatment of myopia, hyperopia and astigmatism. Laser in situ
keratomileusis (LASIK) and photorefractive keratectomy (PRK) are
the most common refractive procedures however others have been
developed in an attempt to overcome complications. These
complications include overcorrection, undercorrection, regression
and stromal opacification among others. A number of common
complications are related to the healing response and have their
roots in the biologic response to surgery. One of the greatest
challenges in corneal biology is to promote tissue repair via
regeneration rather than fibrosis. It is believed that the choice
between regeneration and fibrosis lies in the control of fibroblast
activation [Stramer, et al (2003), Invest Opthalmol Vis Sci; vol
44: 4237-4246, and Fini (1999) Prog Retin Eye Res, vol 18:
529-551]. Cells called myofibroblasts may appear in the
subepithelial stroma 1-2 weeks after surgery or injury.
Myofibroblasts are presumably derived from keratocytes under the
influence of TGF-.beta. [Jester, et al. (2003), Exp Eye Res, vol
77: 581-592]. Corneal haze and stromal scarring are characterized
by reduced corneal transparency and may be associated with
fibroblast and myofibroblast generation. In situ and in vitro
studies have suggested that TGF-.beta. and PDGF are important in
stimulating myofibroblast differentiation [Folger, et al. (2001),
Invest Opthalmol Vis Sci; 42: 2534-2541]. Haze can be noted in the
central interface after LASIK under certain circumstances. These
include diffuse lamellar keratitis, donut-shaped flaps, and
retention of epithelial debris at the interface. It is likely that
each of these is associated with increased access of TGF-.beta.
from epithelial cells to the activated keratocytes [Netto, et al.
(2005), Cornea, vol 24: 509-522]. Regression is most likely due to
heightened epithelial-stromal wound healing interactions such as
increased production of epithelium modulating growth factors by
corneal fibroblasts and or myofibroblasts [Netto, et al. (2005),
above]. Inhibition of TGF-.beta. binding to receptors with topical
anti-TGF-.beta. antibody has been shown to reduce haze induced by
PRK [Jester, et al. (1997), Cornea, vol 16: 177-187]. Given the
known effects of anti-bioactive lipid antibody on the fibrotic
process and TGF-.beta., we believe that it may aid in treating some
of the complications of refractive surgery such as haze, stromal
scarring and regression.
e. Modulation of Glaucoma Filtration Surgery.
Glaucoma is classically thought of a disease whereby elevated
intraocular pressure causes damage to the optic nerve and
ultimately compromises the visual field and or the visual acuity.
Other forms of glaucoma exist where optic nerve damage can occur in
the setting of normal pressure or so called "normal tension
glaucoma". For many patients medications are able to control their
disease, but for others glaucoma filtration surgery is needed
whereby a fistula is surgically created in the eye to allow fluid
to drain. This can be accomplished via trabeculectomy, the
implantation of a medical device or other methods of surgical
intervention. Glaucoma filtration surgery fails due to a wound
healing process characterized by the proliferation of fibroblasts
and ultimately scarring. Anti-metabolites such as 5-fluorouracil
and mitomycin C can reduce subsequent scarring; however, even with
the use of these drugs long term follow up shows that surgical
failure is still a serious clinical problem [Mutsch and Grehn
(2000), Graefes Arch Clin Exp Opthalmol; vol 238: 884-91, and
Fontana, et al. (2006), Opthalmology, vol 113: 930-936]. Studies of
human Tenon's capsule fibroblasts demonstrate that they have the
capacity to synthesize bFGF and PDGF and TGF-.beta. and that these
growth factors are implicated in the tissue repair process after
glaucoma filtration surgery that contributes to the failure of the
procedure. Trpathi, et al. (1996), Exp Eye Res, vol 63: 339-46.
Additional studies have also implicated these growth factors in the
post filtration wound response [Denk, et al. (2003), Curr Eye Res;
vol 27: 35-44] concluded that different isoforms of PDGF are major
stimulators of proliferation of Tenon's capsule fibroblasts after
glaucoma filtration surgery while TGF-.beta. is essential for the
transformation of Tenon's capsule fibroblasts into myofibroblasts.
We have demonstrated that S1P is present in human Tenon's
capsule/conjunctival fibroblasts and that S1P is strongly expressed
in the wound healing response. S1P also stimulates the profibrotic
function of multiple fibroblast cell types and the transformation
into the myofibroblast phenotype and collagen production. Given the
specific pleotropic effects of S1P and its known interactions with
bFGF, PDGF, and TGF-beta, it is believed that an agent that binds,
antagonizes, inhibits the effects or the production of S1P will be
effective at modulating the wound healing and/or fibrotic response
that leads to failure of glaucoma surgery and will be an effective
therapeutic method of enhancing successful surgical outcomes. It is
envisioned that the agent could be administered, e.g., via
intravitreal or subconjunctival injection or topically.
f. Corneal Transplantation.
Corneal transplantation (penetrating keratoplasty (PK)) is the most
successful tissue transplantation procedure in humans. Yet of the
47,000 corneal transplants performed annually in the United States,
corneal allograft rejection is still the leading cause of corneal
graft failure [1 ng, et al. (1998), Opthalmology, vol 105:
1855-1865]. Currently, there is insufficient ability to avert
allograft rejection although immunosuppression and immunomodulation
may be a promising approach. Recently it has been discovered that
CD4(+) T cells function as directly as effector cells and not
helper cells in the rejection of corneal allografts. Hegde, et al.
(2005), Transplantation, vol 79: 23-31. Murine studies have shown
increased numbers of neutrophils, macrophage and mast cells in the
stroma of corneas undergoing rejection. Macrophages were the main
infiltrating cell type followed by T-cells, mast cells and
neutrophils. The early chemokine expression in high risk corneal
transplantation was the mouse homologue of IL-8 (macrophage
inflammatory protein-2) and monocyte chemotactic protein-1 (MCP-1)
[Yamagami, et al. (2005), Mol Vis, vol 11, 632-40].
FTY720 (FTY) is a novel immunosuppressive drug that acts by
altering lymphocyte trafficking; resulting in peripheral blood
lymphopenia and increased lymphocyte counts in lymph nodes. FTY
mediates its immune-modulating effects by binding to some of the
S1P receptors expressed on lymphocytes [Bohler, et al. (2005),
Transplantation, vol 79: 492-5]. The drug is administered orally
and a single oral dose reduced peripheral lymphocyte counts by
30-70%. FTY reduced T-cell subset, CD4(+) cells more than CD8(+)
cells. Bohler, et al. (2004), Nephrol Dial Transplant, vol 19:
702-13. FTY treated mice showed a significant prolongation of
orthotopic corneal-graft survival when administered orally. Zhang,
et al. (2003), Transplantation, vol 76: 1511-3. FTY oral treatment
also significantly delayed rejection and decreased its severity in
a rat-to-mouse model of corneal xenotransplantation [Sedlakova, et
al. (2005), Transplantation, vol 79, 297-303]. Given the known
pathogenesis of allograft rejection combined with the data
suggesting that modulating the effects of the S1P signaling can
improve corneal graft survival, it is believed that immune moieties
that decrease the effective concentration of bioactive lipids,
e.g., SPHINGOMAB, will also be useful in treatment of immunologic
conditions such as allograft rejection, for example by attenuating
the immune response, and thus will likely improve corneal graft
survival after PK. The drug may also has the added advantage that
in addition to systemic administration, local administration, e.g.,
via topical periocular or intraocular delivery, is possible.
Other ocular diseases with an inflammatory or immune component
include chronic vitritis, infections, including herpes simplex,
herpes zoster, and protozoan infections, and ocular
histoplasmosis.
g. Anterior Segment Diseases Characterized by Scarring.
Treatment with an antibody targeted to bioactive lipid also is
believed to benefit several conditions characterized by scarring of
the anterior portion of the eye. These include the following:
i. Trauma
The cornea, as the most anterior structure of the eye, is exposed
to various hazards ranging from airborne debris to blunt trauma
that can result in mechanical trauma. The cornea and anterior
surface of the eye can also be exposed to other forms of trauma
from surgery, and chemical, such as acid and alkali, injuries. The
results of these types of injuries can be devastating often leading
to corneal and conjunctival scarring symblephera formation. In
addition, corneal neovascularization may ensue. Neutrophils
accumulate, their release of leukotrienes, and the presence of
interleukin-1 and interleukin-6, serves to recruit successive waves
of inflammatory cells [Sotozono, et al. (1997), Curr Eye Res, vol
19: 670-676] that infiltrate the cornea and release proteolytic
enzymes, which leads to further damage and break down of corneal
tissue and a corneal melt. In addition, corneal and conjunctival
fibroblasts become activated and invade and leading to collagen
deposition and fibrosis. The undesirable effects of excessive
inflammation and scarring are promoted by TGF-.beta.. Saika, et al.
(2006), Am J Pathol vol 168, 1848-60. This process leads to loss of
corneal transparency and impaired vision. Reduced inflammation,
including decreased neutrophil infiltrates and reduced fibrosis
resulted in faster and more complete healing in a murine model of
alkali burned corneas [Ueno, et al. (2005), Opthalmol Vis Sci, vol
46: 4097-106].
ii. Ocular Cicatricial Pemphigoid (OCP)
OCP is a chronic cicatrizing (scar-forming) autoinimune disease
that primarily affects the conjunctiva. The disease is invariably
progressive and the prognosis is quite poor. In its final stages
conjunctival scarring and the associated keratopathy lead to
bilateral blindness. Histologically the conjunctiva shows
submucosal scarring and chronic inflammation in which mast cell
participation is surprisingly great [Yao, et al. (2003), Ocul
Immunol Inflamm, vol 11: 211-222]. Autoantigens lead to the
formation of autoantibodies. The binding of the autoantibody to the
autoantigen sets in motion a complex series of events with
infiltration of T lymphocytes where CD4 (helper) cells far
outnumber CD8 (suppressor) cells. Macrophage and mast cell
infiltration also ensue as well as the release of proinflammatory
and profibrotic cytokines. Cytokine-induced conjunctival fibroblast
proliferation and activation results, with resultant subepithelial
fibrosis (see examples hereinbelow). Studies have shown a role of
TGF-.beta. and IL-1 in conjunctival fibrosis in patients with OCP
[Razzaque, et al. (2004), Invest Opthalmol Vis Sci, vol 45:
1174-81].
iii. Stevens Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis
(TEN)
SJS and TEN are life-threatening adverse reactions to medications.
The ocular sequelae of these two related conditions can be severe
and involve pathologic changes of the bulbar and palpebral
conjunctiva, eyelids, and cornea. Drugs and infections are the most
common precipitating factors. Chronic eye findings include
scarring, symblepharon formation, and cicatrisation of the
conjunctiva as a result of the initial inflammatory process. This
leads to entropion formation, trichiasis, and instability of the
tear film. Breakdown of the ocular surface leads to corneal
scarring, neovascularization, and in severe cases keratinization.
As in OCP subepithelial fibrosis of the conjunctiva occurs. A
vigorous autoimmune lymphocyte response to a drug or infection is
believed to play a role in development of SJS/TEN. Harilaos, et al.
(2005), Erythema Multiforme, Stevens Johnson Syndrome, and Toxic
Epidermal Necrolysis, in Cornea 2.sup.nd edition. Krachmer, Mannis,
Holland eds. Elesevier Mosby Philadelphia. The infiltrating cell
population in SJS includes macrophages, CD4 positive T cells, and
CD8 positive T cells. This cell population is similar to those seen
in chemical injury. Kawasaki, et al. (2000), J Opthalmol, vol 84:
1191-3.
iv. Pterygium
Clinically a pterygium appears as a fleshy, vascular mass that
occurs in the interpalpebral fissure. The body of the pterygium is
a fleshy fibrovascular mass. Active pterygium is characterized by
marked vascular engorgement and progressive growth. They are firmly
adherent to the globe. In advanced cases the pterygium encroaches
onto the cornea and may cause visual loss secondary to loss of
corneal transparency within the visual axis or irregular
astigmatism. Symptomatically, patients may experience foreign body
sensation, tearing and blurred vision. Histopathology demonstrates
hyalinization of the subepithelial connective tissue of the
substantia propria, increased number of fibroblasts and increased
mast cells. Butrus, et al. (1995), Am J Opthalmol, vol 119:
236-237. Management of pterygium remains problematic. Surgical
excision is often performed however recurrence rates are high
[Krag, et al. (1992), Acta Opthalmol, vol 70: 530]. In order to
help lower the recurrence rate of pterygium, various pharmacologic
adjuvants have been employed such as Mitomycin-C and daunorubicin.
Although these may be helpful, long term data are limited and they
can be associated with scleral thinning and corneal melt.
Dougherty, et al. [(1996), Cornea, vol 15: 537-540, and Lee, et al.
(2001), Cornea, vol 20: 238-42] were the first to demonstrate that
VEGF may play an important role in the development of pterygium and
to identify VEGF and nitric oxide in the epithelium of pterygium.
These workers hypothesized that these as well as other cytokines
are responsible for the fibrovascular ingrowth characteristic of
pterygium. The presence of basic FGF and TGF-beta 1 in both primary
and recurrent pterygium has been demonstrated [Kira, et al. (1998),
Graefes Arch Clin Exp Opthalmol, vol 236: 702-8] and published
morphometric and immunohistochemical evidence further supports the
notion that angiogenesis may play a role in the formation of
pterygium [Marcovich, et al (2002), Curr Eye Res, vol 25:17-22].
Other studies have implicated IL-6 and IL-8 as well as VEGF as
mediators that may be relevant to pterygium development [Di
Girolamo, et al. (2006), Invest Opthalmol Vis Sci, vol 47: 2430-7].
An effective agent against pterygium formation and growth may
diminish the need for surgical intervention or reduce recurrence
rates.
Other ocular diseases and conditions with a fibrogenesis, fibrosis
or scarring component include AMD, diabetic retinopathy,
retinopathy of prematurity, sickle cell retinopathy, ischemic
retinopathy, retinal venous occlusive disease, and contact lens
overwear.
In summary, excessive scarring is an underlying component of the
pathophysiology of many ocular and non-ocular diseases and
conditions. Bioactive lipids like S1P play a role in this process
and an antibody-related treatment to diminish the concentrations of
these agents will likely lead to therapeutic benefit to patients
receiving the treatment. In one embodiment, inhibitors of bioactive
lipids, particularly monoclonal antibodies directed against S1P
and/or it variants, are believed to be useful in modulating
surgical and traumatic wound healing responses.
Fibrosis, Fibrogenesis and Scar Formation:
The formation of subretinal fibrosis leads to irreversible damage
to the photoreceptors and permanent vision loss. As long as the
neovascular complex remains intact, as appears to be the case in
patients treated with anti-VEGF agents, the potential for
subretinal fibrosis and future vision loss persists. In an update
of the PRONTO study of RANIBIZUMAB (Lucentis.RTM.), it was
discovered that those patients who lost vision did so as a result
of either subretinal fibrosis or a RPE tear. An agent that could
diminish the degree of fibroblast infiltration and collagen
deposition would be of value.
Fibroblasts, particularly myofibroblasts, are key cellular elements
in scar formation in response to cellular injury and inflammation
[Tomasek, et al. (2002), Nat Rev Mol Cell Biol, vol 3: 349-63, and
Virag and Murry (2003), Am J Pathol, vol 163: 2433-40]. Collagen
gene expression by myofibroblasts is a hallmark of remodeling and
necessary for scar formation [Sun and Weber (2000), Cardiovasc Res,
vol 46: 250-6, and Sun and Weber (1996), J Mol Cell Cardiol, vol
28: 851-8]. S1P promotes wound healing by activating fibroblast
migration and proliferation while increasing collagen production
[Sun, et al. (1994), J Biol Chem, vol 269: 16512-7]. S1P produced
locally by damaged cells could be responsible for the maladaptive
wound healing associated with remodeling and scar formation. Thus,
it is believed that S1P inhibitors are useful in diseases or
conditions characterized, at least in part, by aberrant
fibrogenesis or fibrosis.
The formation of subretinal fibrosis leads to irreversible damage
to the photoreceptors and permanent vision loss. As long as the
neovascular complex remains intact, as appears to be the case in
patients treated with anti-VEGF agents, the potential for
subretinal fibrosis and future vision loss persists.
Minimizing maladaptive scar formation by neutralization of S1P
could be beneficial and prevent irreversible losses in visual
acuity by limiting the extent of sub-retinal fibrosis and
subsequent photoreceptor damage. Growing evidence suggests that S1P
could contribute to both the early and late stages of maladaptive
retinal remodeling associated with exudative AMD. S1P has a
pronounced non-VEGF dependent pro-angiogenic effect. S1P also
stimulates migration, proliferation and survival of multiple cell
types, including fibroblasts, EC, pericytes and inflammatory
cells--the same cells that participate in the multiple maladaptive
processes of exudative AMD and other ocular disorders. S1P is
linked to the production and activation of VEGF, bFGF, PDGF, and
other growth factors (GFs) implicated in the pathogenesis of
exudative AMD. Finally, S1P may modulate the maturation of naive
vasculature, a process leading to a loss of sensitivity to
anti-angiogenic agents. Inhibiting the action of S1P could be an
effective therapeutic treatment for exudative AMD that may offer
significant advantages over exclusively anti-VEGF approaches or may
act synergistically with them to address the complex processes and
multiple steps that ultimately lead to AMD associated visual
loss.
Currently favored therapeutic modalities for AMD include
Lucentis.RTM. and off-label use of Avastin.RTM. (Genentech, Inc.),
both of which target a single growth factor (VEGF-A) and appear to
exert most of their beneficial effect via an anti-permeability
action resulting in resolution of sub-retinal and intra-retinal
edema, as the actual choroidal neovascular (CNV) lesion does not
markedly involute. However, exudative AMD-related vision loss is
not solely due to CNV-induced sub-retinal and intra-retinal edema.
Pathologic disruption and remodeling of the retinal and subretinal
architecture caused collectively by CNV, sub-retinal fibrosis,
edema and inflammation together result in the loss of visual acuity
associated with AMD. These multiple causes are not addressed by
available treatments, including Lucentis.TM.. Thus a therapeutic
agent that could treat the multiple mechanisms that cause vision
loss would be of great value, either as monotherapy or in
combination with another agent, such as an anti-VEGF agent (e.g.,
Lucentis.RTM. or Avastin.RTM.).
Thus, without wishing to be bound by any particular theory, it is
believed that the level of undesirable sphingolipids such as S1P,
and/or one or more of their metabolites, cause or contribute to the
development of various ocular diseases and disorders where
inappropriate inflammation, fibrosis and/or angiogenesis are
involved in the pathogenesis of the disease. Diseases and
conditions of the eye in which anti-S1P antibodies are likely to be
clinically useful include diabetic retinopathy, retinopathy of
prematurity, diabetic macular edema, PVR, anterior segment diseases
and age-related macular edema, both wet and dry, and after
procedures such as trabeculectomy or valve implantation in
glaucoma.
Anti-S1P Antibodies for the Treatment of Scleroderma
The compositions and methods of the invention will be useful in
treating disorders and diseases characterized, at least in part, by
aberrant neovascularization, angiogenesis, fibrogenesis, fibrosis,
scarring, inflammation, and immune response. One such disease is
scleroderma, which is also referred to as systemic sclerosis.
Scleroderma is an autoimmune disease that causes scarring or
thickening of the skin, and sometimes involves other areas of the
body, including the lungs, heart, and/or kidneys. Scleroderma is
characterized by the formation of scar tissue (fibrosis) in the
skin and organs of the body, which can lead to thickening and
firmness of involved areas, with consequent reduction in function.
Today, about 300,000 Americans have scleroderma, according to the
Scleroderma Foundation. One-third or less of those affected have
widespread disease, while the remaining two-thirds primarily have
skin symptoms. When the disease affects the lungs and causing
scarring, breathing can become restricted because the lungs can no
longer expand as they should. To measure breathing capability,
doctors use a device that assesses forced vital capacity (FVC). In
people with an FVC of less than 50 percent of the expected reading,
the 10-year mortality rate from scleroderma-related lung disease is
about 42 percent. One reason the mortality rate is so high is that
no effective treatment is currently available.
Without wishing to be bound by any particular theory, it is
believed that inappropriate concentrations of lipids such as S1P
and/or its metabolites, cause or contribute to the development of
scleroderma. As such, the compositions and methods of the invention
can be used to treat scleroderma, particularly by decreasing the
effective in vivo concentration of a particular target lipid, for
example, S1P.
As described elsewhere in this application, existing evidence
indicates that S1P is a pro-fibrotic growth factor that can
contribute to fibroblast activation, proliferation, and the
resulting increased fibroblast activity associated with maladaptive
scarring and remodeling. It is believed that S1P bioactivity with
and anti-S1P agent (e.g., an anti-S1P mAb) could indirectly
mitigate the pro-sclerotic effects of PDGF and TGF-.beta..
Moreover, treatment with such an anti-S1P agent could benefit
scleroderma patients by mitigating the direct effects of S1P on
skin and other forms of fibroblasts that contribute to disease
progression.
Cardiovascular and Cerebrovascular Disorders
Without wishing to be bound by any particular theory, the level of
undesirable sphingolipids such as CER, SPH, or S1P, and/or one or
more of their metabolites, may be directly responsible for cardiac
dysfunction, during or after cardiac ischemia such as during
reperfusion injury and the resulting cardiac remodeling and heart
failure.
Because sphingolipids such as S1P are involved in fibrogenesis and
wound healing of liver tissue (Davaille, et al., J. Biol. Chem.
275:34268-34633, 2000; Ikeda, et al., Am J. Physiol. Gastrointest.
Liver Physiol 279:G304-G310, 2000), healing of wounded vasculatures
(Lee, et al., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000),
and other disease states, or events associated with such diseases,
such as cancer, angiogenesis and inflammation (Pyne, et al.,
Biochem. J. 349:385-402, 2000), the compositions and methods of the
disclosure may be applied to treat not only these diseases but
cardiac diseases as well.
This suggests that sphingolipids derived from cardiac or other
non-cerebral sources could contribute to stroke. Consequently,
interfering with sphingolipid production and/or action may be
beneficial in mitigating stroke, particularly in stroke caused by
peripheral vascular disease, atherosclerosis, and cardiac
disorders. Recent evidence suggests that exogenously administered
S1P crosses the blood-brain barrier and promotes cerebral
vasoconstriction (Tosaka, et al., Stroke 32: 2913-2919.2001).
It has been suggested that an early event in the course of cardiac
ischemia (i.e., lack of blood supply to the heart) is an excess
production by the heart muscle of the naturally occurring compound
sphingosine, and that other metabolites, particularly S1P are also
produced either by the heart tissue itself or by components of
blood as a consequence of cardiac sphingolipid production and
subsequent conversion in the blood. The present invention provides
methods for neutralizing S1P with specific humanized monoclonal
antibodies. The present invention thus provides humanized
anti-sphingolipid antibodies and related compositions and methods
to reduce blood and tissue levels of the key sphingolipid, S1P.
Such antibodies are useful, for example, for binding and thus
lowering the effective concentration of, undesirable sphingolipids
in whole blood.
The therapeutic methods and compositions of the invention are said
to be "sphingolipid-based" in order to indicate that these
therapies can change the relative, absolute or available
concentration(s) of certain undesirable, toxic or cardiotoxic
sphingolipids. A "toxic sphingolipid" refers to any sphingolipid
that can cause or enhance the necrosis and/or apoptosis of cells or
otherwise impair function of an organ or tissue (e.g., through
excessive fibrosis), including, in some instances, particular cell
types that are found in specific tissues or organs. "Cardiotoxic
sphingolipids" are toxic sphingolipids that directly or indirectly
promote heart failure through maladaptive scarring (fibrogenesis)
and cause a negative inotropic state or cause or enhance the
necrosis and/or apoptosis of cells found in or associated with the
heart, including but not limited to cardiomyocytes, cardiac neurons
and the like, and/or can cause loss of cardiac function due to the
negative inotropic, arrhythmic coronary vasoconstriction/spasm
effects of the sphingolipids and/or their metabolites. "Undesirable
sphingolipids" include toxic and cardiotoxic sphingolipids, as well
as metabolites, particularly metabolic precursors, of toxic and
cardiotoxic sphingolipids. Undesirable, cardiotoxic, and/or toxic
sphingolipids of particular interest include, but are not limited
to, ceramide (CER), ceraminde-1-phosphate (CIP),
sphingosine-1-phosphate (S-1-P), dihydro-S1P (DHS1P),
sphingosylphosphoryl choline (SPC), sphingosine (SPH;
D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol, or sphinganine)
and various metabolites.
It is known that one of the earliest responses of cardiac myocytes
to hypoxia and reoxygenation is the activation of neutral
sphingomyelinase and the accumulation of ceramide. Hernandez, et
al. (2000), Circ. Res. 86:198-204, 2000. SPH has been allegedly
implicated as mediating an early signaling event in apoptotic cell
death in a variety of cell types (Ohta, et al., FEBS Letters
355:267-270, 1994; Ohta, et al., Cancer Res. 55:691-697, 1995;
Cuvlilier, et al., Nature 381:800-803, 1996). It is postulated that
the cardiotoxic effects of hypoxia may result in part from
sphingolipid production and/or from the inappropriate production of
other metabolites (e.g., protons, calcium, and certain free
radicals) or signaling molecules (e.g., MAP kinases, caspases) that
adversely affect cardiac function.
S1P is stored in platelets and is a normal constituent of human
plasma and serum (Yatomi, et al., J. Biochem. 121:969-973, 1997).
S1P is a coronary vasoconstrictor and has other biological effects
on canine hearts. Sugiyama, et al. (2000), Cardiovascular Res.
46:119-125. A role for S1P in atherosclerosis has been postulated
(Siess, et al., IUBMB Life 49:161-171, 2000). This has been
supported by other data, including evidence that the protective
effect of HDL is due to blocking S1P production (Xia, et al., PNAS
95:14196-14201, 1988; Xia, et al., J Biol Chem 274:33143-33147,
1999).
Sphingomyelin, the metabolic precursor of ceramide, has been
reported to be increased in experimental animals subjected to
hypoxia (Sergeev, et al., Kosm. Biol. Aviakosm. Med. (Russian)
15:71-74, 1981). Other studies have reported that internal
membranes of muscle cells contain high amounts of SPH and
sphingomyelin (Sumnicht, et al., Arch. Biochem. Biophys.
215:628-637, 1982; Sabbadini, et al., Biochem. Biophys. Res. Comm.
193752-758, 1993). Treatment of experimental animals with
fumonisinB fungal toxins result in increase serum levels of SPH and
DHSPH (S1P was not measured) with coincident negative inotropic
effects on the heart (Smithe, et al., Toxicological Sciences
56:240-249, 2000).
Other Diseases or Conditions
Because of the involvement of bioactive lipid signaling in many
processes, including neovascularization, angiogenesis, aberrant
fibrogenesis, fibrosis and scarring, and inflammation and immune
responses, it is believed that inhibitors of these bioactive lipids
will be helpful in a variety of diseases and conditions associated
with one or more of these processes. Such diseases and conditions
may be systemic (e.g., systemic scleroderma) or localized to one or
more specific body systems, parts or organs (e.g., skin, lung,
cardiovascular system or eye).
One way to control the amount of undesirable sphingolipids in a
patient is by providing a composition that comprises one or more
humanized anti-sphingolipid antibodies to bind one or more
sphingolipids, thereby acting as therapeutic "sponges" that reduce
the level of free undesirable sphingolipids. When a compound is
referred to as "free", the compound is not in any way restricted
from reaching the site or sites where it exerts its undesirable
effects. Typically, a free compound is present in blood and tissue,
which either is or contains the site(s) of action of the free
compound, or from which a compound can freely migrate to its
site(s) of action. A free compound may also be available to be
acted upon by any enzyme that converts the compound into an
undesirable compound.
Without wishing to be bound by any particular theory, it is
believed that the level of undesirable sphingolipids such as SPH or
S1P, and/or one or more of their metabolites, cause or contribute
to the development of cardiac and myocardial diseases and
disorders.
Because sphingolipids are also involved in fibrogenesis and wound
healing of liver tissue (Davaille, et al., J. Biol. Chem.
275:34268-34633, 2000; Ikeda, et al., Am J. Physiol. Gastrointest.
Liver Physiol 279:G304-G310, 2000), healing of wounded vasculatures
(Lee, et al., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000),
and other disease states or disorders, or events associated with
such diseases or disorders, such as cancer, angiogenesis, various
ocular diseases associate with excessive fibrosis and inflammation
(Pyne et al., Biochem. J. 349:385-402, 2000), the compositions and
methods of the present disclosure may be applied to treat these
diseases and disorders as well as cardiac and myocardial diseases
and disorders.
One form of sphingolipid-based therapy involves manipulating the
metabolic pathways of sphingolipids in order to decrease the
actual, relative and/or available in vivo concentrations of
undesirable, toxic sphingolipids. The invention provides
compositions and methods for treating or preventing diseases,
disorders or physical trauma, in which humanized anti-sphingolipid
antibodies are administered to a patient to bind undesirable, toxic
sphingolipids, or metabolites thereof.
Such humanized anti-sphingolipid antibodies may be formulated in a
pharmaceutical composition that are useful for a variety of
purposes, including the treatment of diseases, disorders or
physical trauma. Pharmaceutical compositions comprising one or more
humanized anti-sphingolipid antibodies of the invention may be
incorporated into kits and medical devices for such treatment.
Medical devices may be used to administer the pharmaceutical
compositions of the invention to a patient in need thereof, and
according to one embodiment of the invention, kits are provided
that include such devices. Such devices and kits may be designed
for routine administration, including self-administration, of the
pharmaceutical compositions of the invention. Such devices and kits
may also be designed for emergency use, for example, in ambulances
or emergency rooms, or during surgery, or in activities where
injury is possible but where full medical attention may not be
immediately forthcoming (for example, hiking and camping, or combat
situations).
Methods of Administration.
The treatment for diseases and conditions discussed herein can be
achieved by administering agents and compositions of the invention
by various routes employing different formulations and devices.
Suitable pharmaceutically acceptable diluents, carriers, and
excipients are well known in the art. One skilled in the art will
appreciate that the amounts to be administered for any particular
treatment protocol can readily be determined. Suitable amounts
might be expected to fall within the range of 10 .mu.g/dose to 10
g/dose, preferably within 10 mg/dose to 1 g/dose.
Drug substances may be administered by techniques known in the art,
including but not limited to systemic, subcutaneous, intradermal,
mucosal, including by inhalation, and topical administration. The
mucosa refers to the epithelial tissue that lines the internal
cavities of the body. For example, the mucosa comprises the
alimentary canal, including the mouth, esophagus, stomach,
intestines, and anus; the respiratory tract, including the nasal
passages, trachea, bronchi, and lungs; and the genitalia. For the
purpose of this specification, the mucosa also includes the
external surface of the eye, i.e., the cornea and conjunctiva.
Local administration (as opposed to systemic administration) may be
advantageous because this approach can limit potential systemic
side effects, but still allow therapeutic effect.
Pharmaceutical compositions used in the present invention include,
but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
The pharmaceutical formulations used in the present invention may
be prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). Preferred carriers
include those that are pharmaceutically acceptable, particularly
when the composition is intended for therapeutic use in humans. For
non-human therapeutic applications (e.g., in the treatment of
companion animals, livestock, fish, or poultry), veterinarily
acceptable carriers may be employed. In general the formulations
are prepared by uniformly and intimately bringing into association
the active ingredients with liquid carriers or finely divided solid
carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into
any of many possible dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
In one embodiment the pharmaceutical compositions may be formulated
and used as foams. Pharmaceutical foams include formulations such
as, but not limited to, emulsions, microemulsions, creams, jellies,
and liposomes.
While basically similar in nature these formulations vary in the
components and the consistency of the final product. The know-how
on the preparation of such compositions and formulations is
generally known to those skilled in the pharmaceutical and
formulation arts and may be applied to the formulation of the
compositions of the present invention.
In one embodiment, an immune-derived moiety can be delivered to the
eye via, for example, topical drops or ointment, periocular
injection, intracamerally into the anterior chamber or vitreous,
via an implanted depot, or systemically by injection or oral
administration. The quantity of antibody used can be readily
determined by one skilled in the art.
The traditional approaches to delivering therapeutics to the eye
include topical application, redistribution into the eye following
systemic administration or direct intraocular/periocular injections
[Sultana, et al. (2006), Current Drug Delivery, vol 3: 207-217;
Ghate and Edelhauser (2006), Expert Opinion, vol 3: 275-287; and
Kaur and Kanwar (2002), Drug Develop Industrial Pharmacy, vol 28:
473-493]. Anti-S1P or other anti-bioactive lipid antibody
therapeutics would likely be used with any of these approaches
although all have certain perceived advantages and disadvantages.
Topical drops are convenient, but wash away primarily because of
nasolacrimal drainage often delivering less than 5% of the applied
drug into the anterior section of the eye and an even smaller
fraction of that dose to the posterior segment of the globe.
Besides drops, sprays afford another mode for topical
administration. A third mode is ophthalmic ointments or emulsions
can be used to prolong the contact time of the formulation with the
ocular surface although blurring of vision and matting of the
eyelids can be troublesome. Such topical approaches are still
preferable, since systemic administration of therapeutics to treat
ocular disorders exposes the whole body to the potential toxicity
of the drug.
Treatment of the posterior segment of the eye is medically
important because age-related macular degeneration, diabetic
retinopathy, posterior uveitis, and glaucoma are the leading causes
of vision loss in the United States and other developed countries.
Myles, et al. (2005), Adv Drug Deliv Rev; 57: 2063-79. The most
efficient mode of drug delivery to the posterior segment is
intravitreal injection through the pars plana. However, direct
injections require a skilled medical practitioner to effect the
delivery and can cause treatment-limiting anxiety in many patients.
Periocular injections, an approach that includes subconjunctival,
retrobulbar, peribulbar and posterior subtenon injections, are
somewhat less invasive than intravitreal injections. Repeated and
long-term intravitreal injections may cause complications, such as
vitreous hemorrhage, retinal detachment, or endophthalmitis.
The anti-bioactive lipid antibody treatment might also be
administered using one of the newer ocular delivery systems
[Sultana, et al. (2006), Current Drug Delivery, vol 3: 207-217; and
Ghate and Edelhauser (2006), Expert Opinion, vol 3: 275-287],
including sustained or controlled release systems, such as (a)
ocular inserts (soluble, erodible, non-erodible or hydrogel-based),
corneal shields, eg, collagen-based bandage and contact lenses that
provide controlled delivery of drug to the eye, (b) in situ gelling
systems that provide ease of administration as drops that get
converted to gel form in the eye, thereby providing some sustained
effect of drug in the eye, (c) vesicular systems such as liposomes,
niosomes/discomes, etc., that offers advantages of targeted
delivery, bio-compatibility and freedom from blurring of vision,
(d) mucoadhesive systems that provide better retention in the eye,
(e) prodrugs (f) penetration enhancers, (g) lyophilized carrier
systems, (h) particulates, (i) submicron emulsions, (j)
iontophoresis, (k) dendrimers, (l) microspheres including
bioadhesive microspheres, (m) nanospheres and other nanoparticles,
(n) collasomes, and (o) drug delivery systems that combine one or
more of the above stated systems to provide an additive, or even
synergistic, beneficial effect. Most of these approaches target the
anterior segment of the eye and may be beneficial for treating
anterior segment disease. However, one or more of these approaches
still may be useful affecting bioactive lipid concentrations in the
posterior region of the eye because the relatively low molecular
weights of the lipids will likely permit considerable movement of
the lipid within the eye. In addition, the antibody introduced in
the anterior region of the eye may be able to migrate throughout
the eye especially if it is manufactured in a lower weight antibody
variant such as a Fab fragment. Sustained drug delivery systems for
the posterior segment such as those approved or under development
(see references, supra) could also be employed.
As previously mentioned, the treatment of disease of the posterior
retina, choroids, and macula is medically very important. In this
regard, transscleral iontophoresis [Eljarrat-Binstock and Domb
(2006), Control Release, 110: 479-89] is an important advance and
may offer an effective way to deliver antibodies to the posterior
segment of the eye.
Various excipients might also be added to the formulated antibody
to improve performance of the therapy, make the therapy more
convenient or to clearly ensure that the formulated antibody is
used only for its intended, approved purpose. Examples of
excipients include chemicals to control pH, antimicrobial agents,
preservatives to prevent loss of antibody potency, dyes to identify
the formulation for ocular use only, solubilizing agents to
increase the concentration of antibody in the formulation,
penetration enhancers and the use of agents to adjust isotonicity
and/or viscosity. Inhibitors of, e.g., proteases, could be added to
prolong the half life of the antibody. In one embodiment, the
antibody is delivered to the eye by intravitreal injection in a
solution comprising phosphate-buffered saline at a suitable pH for
the eye.
The anti-S1P agent (e.g., a humanized antibody) can also be
chemically modified to yield a pro-drug that is administered in one
of the formulations or devices previously described above. The
active form of the antibody is then released by action of an
endogenous enzyme. Possible ocular enzymes to be considered in this
application are the various cytochrome p450s, aldehyde reductases,
ketone reductases, esterases or N-acetyl-.beta.-glucosamidases.
Other chemical modifications to the antibody could increase its
molecular weight, and as a result, increase the residence time of
the antibody in the eye. An example of such a chemical modification
is pegylation [Harris and Chess (2003), Nat Rev Drug Discov; 2:
214-21], a process that can be general or specific for a functional
group such as disulfide [Shaunak, et al. (2006), Nat Chem Biol;
2:312-3] or a thiol [Doherty, et al. (2005), Bioconjug Chem; 16:
1291-8].
The examples herein below describe the production of humanized and
variant anti-sphingolipid antibodies with desirable properties from
a therapeutic perspective, including strong binding affinity for
sphingolipids. In particular, the invention is drawn to S1P and its
variants which may include S1P itself defined as
sphingosine-1-phosphate [sphingene-1-phosphate;
D-erythro-sphingosine-1-phosphate; sphing-4-enine-1-phosphate;
(E,2S,3R)-2-amino-3-hydroxy-octadec-4-enoxy]phosphonic acid (AS
26993-30-6), DHS1P is defined as dihydrosphingosine-1-phosphate
[sphinganine-1-phosphate;
[(2S,3R)-2-amino-3-hydroxy-octadecoxy]phosphonic acid;
D-Erythro-dihydro-D-sphingosine-1-phosphate (CAS 19794-97-9]; SPC
is sphingosylphosphoryl choline, lysosphingomyelin,
sphingosylphosphocholine, sphingosine phosphorylcholine,
ethanaminium;
2-((((2-amino-3-hydroxy-4-octadecenyl)oxy)hydroxyphosphinyl)oxy)-N,N,N-tr-
imethyl-, chloride, (R-(R*,S*-(E))),
2-[[(E,2R,3S)-2-amino-3-hydroxy-octadec-4-enoxy]-hydroxy-phosphoryl]oxyet-
hyl 1-trimethyl-azanium chloride (CAS 10216-23-6).
Antibody Generation and Characterization
Antibody affinities may be determined as described in the examples
herein below. Preferred humanized or variant antibodies are those
which bind a sphingolipid with a K.sub.d value of no more than
about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 M, and most preferably no more than about
5.times.10.sup.-9 M.
Aside from antibodies with strong binding affinity for
sphingolipids, it is also desirable to select humanized or variant
antibodies that have other beneficial properties from a therapeutic
perspective. For example, the antibody may be one that reduce
angiogenesis and alter tumor progression. Preferably, the antibody
has an effective concentration 50 (EC50) value of no more than
about 10 ug/ml, preferably no more than about 1 ug/ml, and most
preferably no more than about 0.1 ug/ml, as measured in a direct
binding ELISA assay. Preferably, the antibody has an effective
concentration value of no more than about 10 ug/ml, preferably no
more than about 1 ug/ml, and most preferably no more than about 0.1
ug/ml, as measured in cell assays in presence of 1 uM of S1P, for
example, at these concentrations the antibody is able to inhibit
sphingolipid-induced IL-8 release in vitro by at least 10%.
Preferably, the antibody has an effective concentration value of no
more than about 10 ug/ml, preferably no more than about 1 ug/ml,
and most preferably no more than about 0.1 ug/ml, as measured in
the CNV animal model after laser burn, for example, at these
concentrations the antibody is able to inhibit sphingolipid-induced
neovascularization in vivo by at least 50%.
Assays for determining the activity of the anti-sphingolipid
antibodies of the invention include ELISA assays as shown in the
examples hereinbelow.
Preferably the humanized or variant antibody fails to elicit an
immunogenic response upon administration of a therapeutically
effective amount of the antibody to a human patient. If an
immunogenic response is elicited, preferably the response will be
such that the antibody still provides a therapeutic benefit to the
patient treated therewith.
According to one embodiment of the invention, humanized
anti-sphingolipid antibodies bind the "epitope" as herein defined.
To screen for antibodies that bind to the epitope on a sphingolipid
bound by an antibody of interest (e.g., those that block binding of
the antibody to sphingolipid), a routine cross-blocking assay such
as that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, epitope mapping, e.g., as described in
Champe, et al. [J. Biol. Chem. 270:1388-1394 (1995)], can be
performed to determine whether the antibody binds an epitope of
interest.
The antibodies of the invention have a heavy chain variable domain
comprising an amino acid sequence represented by the formula:
FR1-CDRHI-FR2-CDRH2-FR3-CDRH3-FR4, wherein "FR1-4" represents the
four framework regions and "CDRHI-3" represents the three
hypervariable regions of an anti-sphingolipid antibody variable
heavy domain. FR1-4 may be derived from a "consensus sequence" (for
example the most common amino acids of a class, subclass or
subgroup of heavy or light chains of human immunoglobulins) as in
the examples below or may be derived from an individual human
antibody framework region or from a combination of different
framework region sequences. Many human antibody framework region
sequences are compiled in Kabat, et al., supra, for example. In one
embodiment, the variable heavy FR is provided by a consensus
sequence of a human immunoglobulin subgroup as compiled by Kabat,
et al., above. Preferably, the human immunoglobulin subgroup is
human heavy chain subgroup III (e.g., as in SEQ ID NO:16).
The human variable heavy FR sequence preferably has one or more
substitutions therein, e.g., wherein the human FR residue is
replaced by a corresponding nonhuman residue (by "corresponding
nonhuman residue" is meant the nonhuman residue with the same Kabat
positional numbering as the human residue of interest when the
human and nonhuman sequences are aligned), but replacement with the
nonhuman residue is not necessary. For example, a replacement FR
residue other than the corresponding nonhuman residue can be
selected by phage display. Exemplary variable heavy FR residues
which may be substituted include any one or more of FR residue
numbers: 37H, 49H, 67H, 69H, 71H, 73H, 75H, 76H, 78H, and 94H
(Kabat residue numbering employed here). Preferably at least two,
or at least three, or at least four of these residues are
substituted. A particularly preferred combination of FR
substitutions is: 49H, 69H, 71H, 73H, 76H, 78H, and 94H. With
respect to the heavy chain hypervariable regions, these preferably
have amino acid sequences listed in Table 2, below.
The antibodies of the preferred embodiment herein have a light
chain variable domain comprising an amino acid sequence represented
by the formula: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4, wherein "FR1-4"
represents the four framework regions and "CDRL1-3" represents the
three hypervariable regions of an anti-sphingolipid antibody
variable heavy domain. FR1-4 may be derived from a "consensus
sequence" (for example, the most common amino acids of a class,
subclass or subgroup of heavy or light chains of human
immunoglobulins) as in the examples below or may be derived from an
individual human antibody framework region or from a combination of
different framework region sequences. In one preferred embodiment,
the variable light FR is provided by a consensus sequence of a
human immunoglobulin subgroup as compiled by Kabat, et al., above.
Preferably, the human immunoglobulin subgroup is human kappa light
chains subgroup I (e.g., as in SEQ ID NO: 17).
The human variable light FR sequence preferably has substitutions
therein, e.g., wherein a human FR residue is replaced by a
corresponding mouse residue, but replacement with the nonhuman
residue is not necessary. For example, a replacement residue other
than the corresponding nonhuman residue may be selected by phage
display. Exemplary variable light FR residues that may be
substituted include any one or more of FR residue numbers,
including, but not limited to, F4, Y36, Y49, G64, S67.
With respect to the CDRs, these preferably have amino acid
sequences listed in Table 2, below.
Methods for generating humanized anti-sphingolipid antibodies of
interest herein are elaborated in more detail below.
A. Antibody Preparation
Methods for humanizing nonhuman anti-sphingolipid antibodies and
generating variants of anti-sphingolipid antibodies are described
in the Examples below. In order to humanize an anti-sphingolipid
antibody, the nonhuman antibody starting material is prepared.
Where a variant is to be generated, the parent antibody is
prepared. Exemplary techniques for generating such nonhuman
antibody starting material and parent antibodies will be described
in the following sections.
(i) Antigen Preparation.
The sphingolipid antigen to be used for production of antibodies
may be, e.g., intact sphingolipid or a portion of a sphingolipid
(e.g., a sphingolipid fragment comprising an "epitope"). Other
forms of antigens useful for generating antibodies will be apparent
to those skilled in the art. The sphingolipid antigen used to
generate the antibody, is described in the examples below. In one
embodiment, the antigen is a derivatized form of the sphingolipid,
and may be associated with a carrier protein.
(ii) Polyclonal Antibodies.
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the
relevant antigen and an adjuvant. It may be useful to conjugate the
relevant antigen to a protein that is immunogenic in the species to
be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates,
or derivatives by combining, e.g., 100 ug or 5 ug of the protein or
conjugate (for rabbits or mice, respectively) with three volumes of
Freund's complete adjuvant and injecting the solution intradermally
at multiple sites. One month later the animals are boosted with 0.1
to 0.2 times the original amount of peptide or conjugate in
Freund's complete adjuvant by subcutaneous injection at multiple
sites. Seven to 14 days later the animals are bled and the serum is
assayed for antibody titer. Animals are boosted until the titer
plateaus. Preferably, the animal is boosted with the conjugate of
the same antigen, but conjugated to a different protein and/or
through a different cross-linking reagent. Conjugates also can be
made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum may be suitably used to enhance the
immune response.
(iii) Monoclonal Antibodies.
Monoclonal antibodies may be made using the hybridoma method first
described by Kohler, et al., Nature, 256:495 (1975), or by other
suitable methods, including by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other
appropriate host animal, such as a hamster or macaque monkey, is
immunized as hereinabove described to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support
stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOP-21 and M.C.-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur, et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies
produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbant assay (ELISA).
The binding affinity of a monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson, et al., Anal.
Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones may be
subcloned by limiting dilution procedures and grown by standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
pp. 59-103 (Academic Press, 1986)). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably
separated from the culture medium, ascites fluid, or serum by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
(iv) Humanization and Amino Acid Sequence Variants.
Example 12, below, describes procedures for humanization of an
anti-sphingolipid antibody. General methods for humanization are
described in, for example, U.S. Pat. No. 5,861,155, US19960652558,
U.S. Pat. No. 6,479,284, US20000660169, U.S. Pat. No. 6,407,213,
US19930146206, U.S. Pat. No. 6,639,055, US20000705686, U.S. Pat.
No. 6,500,931, US19950435516, U.S. Pat. Nos. 5,530,101, 5,585,089,
US19950477728, U.S. Pat. No. 5,693,761, US19950474040, U.S. Pat.
No. 5,693,762, US19950487200, U.S. Pat. No. 6,180,370,
US19950484537, US2003229208, US20030389155, U.S. Pat. No.
5,714,350, US19950372262, U.S. Pat. No. 6,350,861, US19970862871,
U.S. Pat. No. 5,777,085, US19950458516, U.S. Pat. No. 5,834,597,
US19960656586, U.S. Pat. No. 5,882,644, US19960621751, U.S. Pat.
No. 5,932,448, US19910801798, U.S. Pat. No. 6,013,256,
US19970934841, U.S. Pat. No. 6,129,914, US19950397411, U.S. Pat.
Nos. 6,210,671, 6,329,511, US19990450520, US2003166871,
US20020078757, U.S. Pat. No. 5,225,539, US19910782717, U.S. Pat.
No. 6,548,640, US19950452462, U.S. Pat. No. 5,624,821, and
US19950479752. In certain embodiments, it may be desirable to
generate amino acid sequence variants of these humanized
antibodies, particularly where these improve the binding affinity
or other biological properties of the humanized antibody. Example
12 describes methodologies for generating amino acid sequence
variants of an anti-sphingolipid antibody with enhanced affinity
relative to the parent antibody.
Amino acid sequence variants of the anti-sphingolipid antibody are
prepared by introducing appropriate nucleotide changes into the
anti-sphingolipid antibody DNA, or by peptide synthesis. Such
variants include, for example, deletions from, and/or insertions
into and/or substitutions of, residues within the amino acid
sequences of the anti-sphingolipid antibodies of the examples
herein. Any combination of deletion, insertion, and substitution is
made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
humanized or variant anti-sphingolipid antibody, such as changing
the number or position of glycosylation sites.
A useful method for identification of certain residues or regions
of the anti-sphingolipid antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis," as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
sphingolipid antigen. Those amino acid locations demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at, or for, the sites of
substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per
se need not be predetermined. For example, to analyze the
performance of a mutation at a given site, ala scanning or random
mutagenesis is conducted at the target codon or region and the
expressed anti-sphingolipid antibody variants are screened for the
desired activity. Amino acid sequence insertions include amino-
and/or carboxyl-terminal fusions ranging in length from one residue
to polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-sphingolipid
antibody with an N-terminal methionyl residue or the antibody fused
to an epitope tag. Other insertional variants of the
anti-sphingolipid antibody molecule include the fusion to the N- or
C-terminus of the anti-sphingolipid antibody of an enzyme or a
polypeptide which increases the serum half-life of the
antibody.
Another type of variant is an amino acid substitution variant.
These variants have at least one amino acid residue in the
anti-sphingolipid antibody molecule removed and a different residue
inserted in its place. The sites of greatest interest for
substitutional mutagenesis include the hypervariable regions, but
FR alterations are also contemplated. Conservative substitutions
are preferred substitutions. If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary" substitutions listed below, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00001 TABLE 1 Exemplary Amino Acid Residue Substitutions
Amino acid residue (symbol) Exemplary substitutions Ala (A) val;
leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys;
gln arg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu
asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; arg
arg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L)
norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro
(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;
norleucine
Substantial modifications in the biological properties of the
antibody are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring residues are divided into
groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.
Any cysteine residue not involved in maintaining the proper
conformation of the humanized or variant anti-sphingolipid antibody
also may be substituted, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
One type of substitutional variant involves substituting one or
more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants is affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g., 6-7 sites) are mutated to
generate all possible amino substitutions at each site. The
antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene
IIII product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or in addition, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and sphingolipid. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
Another type of amino acid variant of the antibody alters the
original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
Glycosylation of antibodies is typically either N-linked and/or or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the most common recognition
sequences for enzymatic attachment of the carbohydrate moiety to
the asparagine side chain. Thus, the presence of either of these
tripeptide sequences in a polypeptide creates a potential
glycosylation site. O-linked glycosylation refers to the attachment
of one of the sugars N-aceylgalactosamine, galactose, or xylose to
a hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites). The alteration may also be made
by the addition of, or substitution by, one or more serine or
threonine residues to the sequence of the original antibody (for
O-linked glycosylation sites).
Nucleic acid molecules encoding amino acid sequence variants of the
anti-sphingolipid antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-sphingolipid
antibody.
(v) Human Antibodies.
As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits, et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits, et al.,
Nature, 362:255-258 (1993); Bruggermann, et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
Human antibodies can also be derived from phage-display libraries
(Hoogenboom, et al., J. Mol. Biol., 227:381 (1991); Marks, et al.,
J. Mol. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and
5,573,905). As discussed above, human antibodies may also be
generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos.
5,567,610 and 5,229,275) or by other suitable methods.
(vi) Antibody Fragments.
In certain embodiments, the humanized or variant anti-sphingolipid
antibody is an antibody fragment. Various techniques have been
developed for the production of antibody fragments. Traditionally,
these fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto, et al., Journal of Biochemical and
Biophysical Methods 24:107-117 (1992); and Brennan, et al., Science
229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. For example, Fab'-SH fragments
can be directly recovered from E. coli and chemically coupled to
form F(ab').sub.2 fragments (Carter, et al., Bio/Technology
10:163-167 (1992)). In another embodiment, the F(ab').sub.2 is
formed using the leucine zipper GCN4 to promote assembly of the
F(ab').sub.2 molecule. According to another approach, Fv, Fab or
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner.
(vii) Multispecific Antibodies.
In some embodiments, it may be desirable to generate multispecific
(e.g., bispecific) humanized or variant anti-sphingolipid
antibodies having binding specificities for at least two different
epitopes. Exemplary bispecific antibodies may bind to two different
epitopes of the sphingolipid. Alternatively, an anti-sphingolipid
arm may be combined with an arm which binds to a different
molecule. Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies).
According to another approach for making bispecific antibodies, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers that are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the C.sub.H3 domain of an antibody constant domain.
In this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g., tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g., alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers. See, e.g., U.S. Pat. No. 5,731,168.
Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For example, one of the antibodies in the
heteroconjugate can be coupled to avidin, the other to biotin.
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in, for example, U.S. Pat. No.
4,676,980, along with a number of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody
fragments have also been described in the literature. For example,
bispecific antibodies can be prepared using chemical linkage.
Brennan, et al., Science 229:81 (1985) describe a procedure wherein
intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes. In yet a further embodiment,
Fab'-SH fragments directly recovered from E. coli can be chemically
coupled in vitro to form bispecific antibodies. Shalaby, et al., J.
Exp. Med. 175:217-225 (1992).
Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced
using leucine zippers. Kostelny, et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger, et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker that is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, e.g., Gruber, et al., J.
Immunol. 152:5368 (1994). Alternatively, the bispecific antibody
may be a "linear antibody" produced as described in, fror example,
Zapata, et al. Protein Eng. 8(10): 1057-1062 (1995).
Antibodies with more than two valencies are also contemplated. For
example, trispecific antibodies can be prepared. Tutt et al., J.
Immunol. 147:60 (1991).
An antibody (or polymer or polypeptide) of the invention comprising
one or more binding sites per arm or fragment thereof will be
referred to herein as "multivalent" antibody. For example a
"bivalent" antibody of the invention comprises two binding sites
per Fab or fragment thereof whereas a "trivalent"polypeptide of the
invention comprises three binding sites per Fab or fragment
thereof. In a multivalent polymer of the invention, the two or more
binding sites per Fab may be binding to the same or different
antigens. For example, the two or more binding sites in a
multivalent polypeptide of the invention may be directed against
the same antigen, for example against the same parts or epitopes of
said antigen or against two or more same or different parts or
epitopes of said antigen; and/or may be directed against different
antigens; or a combination thereof. Thus, a bivalent polypeptide of
the invention for example may comprise two identical binding sites,
may comprise a first binding sites directed against a first part or
epitope of an antigen and a second binding site directed against
the same part or epitope of said antigen or against another part or
epitope of said antigen; or may comprise a first binding sites
directed against a first part or epitope of an antigen and a second
binding site directed against the a different antigen. However, as
will be clear from the description hereinabove, the invention is
not limited thereto, in the sense that a multivalent polypeptide of
the invention may comprise any number of binding sites directed
against the same or different antigens.
An antibody (or polymer or polypeptide) of the invention that
contains at least two binding sites per Fab or fragment thereof, in
which at least one binding site is directed against a first antigen
and a second binding site directed against a second antigen
different from the first antigen, will also be referred to as
"multispecific". Thus, a "bispecific" polymer comprises at least
one site directed against a first antigen and at least one a second
site directed against a second antigen, whereas a "trispecific" is
a polymer that comprises at least one binding site directed against
a first antigen, at least one further binding site directed against
a second antigen, and at least one further binding site directed
against a third antigen, etc. Accordingly, in their simplest form,
a bispecific polypeptide of the invention is a bivalent polypeptide
(per Fab) of the invention. However, as will be clear from the
description hereinabove, the invention is not limited thereto, in
the sense that a multispecific polypeptide of the invention may
comprise any number of binding sites directed against two or more
different antigens.
(viii) Other Modifications.
Other modifications of the humanized or variant anti-sphingolipid
antibody are contemplated. For example, the invention also pertains
to immunoconjugates comprising the antibody described herein
conjugated to a cytotoxic agent such as a toxin (e.g., an
enzymatically active toxin of bacterial, fungal, plant or animal
origin, or fragments thereof), or a radioactive isotope (for
example, a radioconjugate). Conjugates are made using a variety of
bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
The anti-sphingolipid antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang,
et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.
Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation
time are disclosed in U.S. Pat. No. 5,013,556. For example,
liposomes can be generated by the reverse phase evaporation method
with a lipid composition comprising phosphatidyl choline,
cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
Liposomes are extruded through filters of defined pore size to
yield liposomes with the desired diameter. Fab' fragments of the
antibody of the present invention can be conjugated to the
liposomes as described in Martin, et al., J. Biol. Chem.
257:286-288 (1982) via a disulfide interchange reaction. Another
active ingredient is optionally contained within the liposome.
Enzymes or other polypeptides can be covalently bound to the
anti-sphingolipid antibodies by techniques well known in the art
such as the use of the heterobifunctional crosslinking reagents
discussed above. Alternatively, fusion proteins comprising at least
the antigen binding region of an antibody of the invention linked
to at least a functionally active portion of an enzyme of the
invention can be constructed using recombinant DNA techniques well
known in the art (see, e.g., Neuberger, et al., Nature 312:604-608
(1984)).
In certain embodiments of the invention, it may be desirable to use
an antibody fragment, rather than an intact antibody, to increase
penetration of target tissues and cells, for example. In this case,
it may be desirable to modify the antibody fragment in order to
increase its serum half life. This may be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g., by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis). See,
e.g., U.S. Pat. No. 6,096,871.
Covalent modifications of the humanized or variant
anti-sphingolipid antibody are also included within the scope of
this invention. They may be made by chemical synthesis or by
enzymatic or chemical cleavage of the antibody, if applicable.
Other types of covalent modifications of the antibody are
introduced into the molecule by reacting targeted amino acid
residues of the antibody with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues. Exemplary covalent modifications of
polypeptides are described in U.S. Pat. No. 5,534,615, specifically
incorporated herein by reference. A preferred type of covalent
modification of the antibody comprises linking the antibody to one
of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol, polypropylene glycol, or polyoxyalkylenes, in the manner
set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
B. Vectors, Host Cells and Recombinant Methods
The invention also provides isolated nucleic acid encoding the
humanized or variant anti-sphingolipid antibody, vectors and host
cells comprising the nucleic acid, and recombinant techniques for
the production of the antibody.
For recombinant production of the antibody, the nucleic acid
encoding it may be isolated and inserted into a replicable vector
for further cloning (amplification of the DNA) or for expression.
In another embodiment, the antibody may be produced by homologous
recombination, e.g., as described in U.S. Pat. No. 5,204,244. DNA
encoding the monoclonal antibody is readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of the antibody). Many vectors are
available. The vector components generally include, but are not
limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence, as
described, for example, in U.S. Pat. No. 5,534,615.
Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P), Pseudomonas such as P.
aeruginosa, and Streptomyces. One preferred E. coli cloning host is
E. coli 294 (ATCC 31,446), although other strains such as E. coli
B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are
suitable. These examples are illustrative rather than limiting.
In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable cloning or expression hosts for
anti-sphingolipid antibody-encoding vectors. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used
among lower eukaryotic host microorganisms. However, a number of
other genera, species, and strains are commonly available and
useful herein, such as Schizosaccharomyces pombe; Kluyveromyces
hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,
and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP
183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora
crassa; Schwanniomyces such as Schwanniomyces occidentalis; and
filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans and A.
niger.
Suitable host cells for the expression of glycosylated
anti-sphingolipid antibodies are derived from
multicellularorganisms. Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham, et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub, et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TR1 cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-68
(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep
G2).
Host cells are transformed with the above-described expression or
cloning vectors for anti-sphingolipid antibody production and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
The host cells used to produce the anti-sphingolipid antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham, et al., Meth. Enz.
58:44 (1979), Barnes, et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. If the antibody is produced intracellularly, as a
first step, the particulate debris, either host cells or lysed
fragments, is removed, for example, by centrifugation or
ultrafiltration. Carter, et al., Bio/Technology 10:163-167 (1992)
describe a procedure for isolating antibodies that are secreted to
the periplasmic space of E. coli. Briefly, cell paste is thawed in
the presence of sodium acetate (pH 3.5), EDTA, and
phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris
can be removed by centrifugation. Where the antibody is secreted
into the medium, supernatants from such expression systems are
generally first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
The antibody composition prepared from the cells can be purified
using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human heavy chains (Lindmark, et al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for human .gamma.3 (Guss, et al., EMBO J. 5:15671575
(1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM. resin
(J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification, such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.,
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
C. Pharmaceutical Formulations
Therapeutic formulations of an antibody or immune-derived moiety of
the invention are prepared for storage by mixing the antibody
having the desired degree of purity with optional physiologically
acceptable carriers, excipients, or stabilizers (see, e.g.,
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.
(1980).
The formulations to be used for in vivo administration must be
sterile. This is readily accomplished for instance by filtration
through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, or microcapsule.
Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919),
copolymers of L-glutamic acid and .gamma.-ethyl-L-glutamate,
non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the Lupron Depot.TM.
(injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric
acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
D. Non-Therapeutic Uses for the Antibodies
The antibodies of the invention may be used as affinity
purification agents. In this process, the antibodies are
immobilized on a solid phase such a Sephadex resin or filter paper,
using methods well known in the art. The immobilized antibody is
contacted with a sample containing the sphingolipid to be purified,
and thereafter the support is washed with a suitable solvent that
will remove substantially all the material in the sample except the
sphingolipid, which is bound to the immobilized antibody. Finally,
the support is washed with another suitable solvent, such as
glycine buffer, for instance between pH 3 to pH 5.0, that will
release the sphingolipid from the antibody.
Anti-sphingolipid antibodies may also be useful in diagnostic
assays for sphingolipid, e.g., detecting its expression in specific
cells, tissues (such as biopsy samples), or bodily fluids. Such
diagnostic methods may be useful in diagnosis of a cardiovascular
or cerebrovascular disease or disorder.
For diagnostic applications, the antibody typically will be labeled
with a detectable moiety. Numerous labels are available which can
be generally grouped into the following categories:
(a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I, .sup.3H,
and .sup.131I. The antibody can be labeled with the radioisotope
using the techniques described in Current Protocols in Immunology,
Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York,
N.Y., Pubs. (1991), for example, and radioactivity can be measured
using scintillation counting.
(b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
(c) Various enzyme-substrate labels are available. For example,
U.S. Pat. No. 4,275,149 provides a review of some of these. The
enzyme generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light that can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan, et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73:147-166 (1981).
Examples of enzyme-substrate combinations include, for example:
(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a
substrate, wherein the hydrogen peroxidase oxidizes a dye precursor
(e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl
benzidine hydrochloride (TMB));
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as
chromogenic substrate; and (iii) .beta.-D-galactosidase
(.beta.-D-Gal) with a chromogenic substrate (e.g.,
p-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
Numerous other enzyme-substrate combinations are available to those
skilled in the art. For a general review of these, see U.S. Pat.
Nos. 4,275,149 and 4,318,980.
Sometimes, the label is indirectly conjugated with the antibody.
The skilled artisan will be aware of various techniques for
achieving this. For example, the antibody can be conjugated with
biotin and any of the three broad categories of labels mentioned
above can be conjugated with avidin, or vice versa. Biotin binds
selectively to avidin and thus, the label can be conjugated with
the antibody in this indirect manner. Alternatively, to achieve
indirect conjugation of the label with the antibody, the antibody
is conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above is conjugated with an
anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect
conjugation of the label with the antibody can be achieved.
In another embodiment of the invention, the anti-sphingolipid
antibody need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the
anti-sphingolipid antibody.
The antibodies of the present invention may be employed in any
known assay method, such as competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays. See,
e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp.
147-158 (CRC Press, Inc. 1987).
Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of sphingolipid in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insoluble before or after the competition, so that the standard and
analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte that remain unbound.
Sandwich assays involve the use of two antibodies, each capable of
binding to a different immunogenic portion, or epitope, of the
protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody that is immobilized on a solid
support, and thereafter a second antibody binds to the analyte,
thus forming an insoluble three-part complex. See, e.g., U.S. Pat.
No. 4,376,110. The second antibody may itself be labeled with a
detectable moiety (direct sandwich assays) or may be measured using
an anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assay). For example, one type of sandwich
assay is an ELISA assay, in which case the detectable moiety is an
enzyme.
For immunohistochemistry, the blood or tissue sample may be fresh
or frozen or may be embedded in paraffin and fixed with a
preservative such as formalin, for example.
The antibodies may also be used for in vivo diagnostic assays.
Generally, the antibody is labeled with a radionuclide (such as
.sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I, .sup.3H,
.sup.32P, or .sup.35S) so that the bound target molecule can be
localized using immunoscintillography.
E. Diagnostic Kits
As a matter of convenience, the antibody of the present invention
can be provided in a kit, for example, a packaged combination of
reagents in predetermined amounts with instructions for performing
the diagnostic assay. Where the antibody is labeled with an enzyme,
the kit will include substrates and cofactors required by the
enzyme (e.g., a substrate precursor which provides the detectable
chromophore or fluorophore). In addition, other additives may be
included such as stabilizers, buffers (e.g., a block buffer or
lysis buffer) and the like. The relative amounts of the various
reagents may be varied widely to provide for concentrations in
solution of the reagents which substantially optimize the
sensitivity of the assay. Particularly, the reagents may be
provided as dry powders, usually lyophilized, including excipients
which on dissolution will provide a reagent solution having the
appropriate concentration.
F. Therapeutic Uses for the Antibody
For therapeutic applications, the anti-sphingolipid antibodies of
the invention are administered to a mammal, preferably a human, in
a pharmaceutically acceptable dosage form such as those discussed
above, including those that may be administered to a human
intravenously as a bolus or by continuous infusion over a period of
time, by intramuscular, intraperitoneal, intra-cerebrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes.
For the prevention or treatment of disease, the appropriate dosage
of antibody will depend on the type of disease to be treated, as
defined above, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments.
Depending on the type and severity of the disease, about 1 ug/kg to
about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. A typical daily or weekly dosage might range from about 1
.mu.g/kg to about 20 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment is repeated until
a desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful. The progress of this therapy is
easily monitored by conventional techniques and assays, including,
for example, radiographic imaging.
According to another embodiment of the invention, the effectiveness
of the antibody in preventing or treating disease may be improved
by administering the antibody serially or in combination with
another agent that is effective for those purposes, such as
chemotherapeutic anti-cancer drugs, for example. Such other agents
may be present in the composition being administered or may be
administered separately. The antibody is suitably administered
serially or in combination with the other agent.
G. Articles of Manufacture
In another embodiment of the invention, an article of manufacture
containing materials useful for the treatment of the disorders
described above is provided. The article of manufacture comprises a
container and a label. Suitable containers include, for example,
bottles, vials, syringes, and test tubes. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agent in the composition is the anti-sphingolipid antibody. The
label on, or associated with, the container indicates that the
composition is used for treating the condition of choice. The
article of manufacture may further comprise a second container
comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
use.
The invention will be better understood by reference to the
following Examples, which are intended to merely illustrate the
best mode now known for practicing the invention. The scope of the
invention is not to be considered limited thereto.
EXAMPLES
Example 1
Murine Monoclonal Antibody to S1P (Sphinsomab.TM.; LT1002)
One type of therapeutic antibody specifically binds undesirable
sphingolipids to achieve beneficial effects such as, e.g., (1)
lowering the effective concentration of undesirable, toxic
sphingolipids (and/or the concentration of their metabolic
precursors) that would promote an undesirable effect such as a
cardiotoxic, tumorigenic, or angiogenic effect; (2) to inhibit the
binding of an undesirable, toxic, tumorigenic, or angiogenic
sphingolipids to a cellular receptor therefore, and/or to lower the
concentration of a sphingolipid that is available for binding to
such a receptor. Examples of such therapeutic effects include, but
are not limited to, the use of anti-S1P antibodies to lower the
effective in vivo serum concentration of available S1P, thereby
blocking or at least limiting S1P's tumorigenic and angiogenic
effects and its role in post-MI heart failure, cancer, or
fibrongenic diseases.
Thiolated S1P was synthesized to contain a reactive group capable
of cross-linking the essential structural features of S1P to a
carrier molecule such as KLH. Prior to immunization, the thio-S1P
analog was conjugated via IOA or SMCC cross-linking to protein
carriers (e.g., KLH) using standard protocols. SMCC is a
heterobifunctional crosslinker that reacts with primary amines and
sulfhydryl groups, and represents a preferred crosslinker.
Swiss Webster or BALB-C mice were immunized four times over a two
month period with 50 .mu.g of immunogen (SMCC facilitated conjugate
of thiolated-S1P and KLH) per injection. Serum samples were
collected two weeks after the second, third, and fourth
immunizations and screened by direct ELISA for the presence of
anti-S1P antibodies. Spleens from animals that displayed high
titers of the antibody were subsequently used to generate
hybridomas per standard fusion procedures. The resulting hybridomas
were grown to confluency, after which the cell supernatant was
collected for ELISA analysis. Of the 55 mice that were immunized, 8
were good responders, showing significant serum titers of
antibodies reactive to S1P. Fusions were subsequently carried out
using the spleens of these mice and myeloma cells according to
established procedures. The resulting 1,500 hybridomas were then
screened by direct ELISA, yielding 287 positive hybridomas. Of
these 287 hybridomas screened by direct ELISA, 159 showed
significant titers. Each of the 159 hybridomas was then expanded
into 24-well plates. The cell-conditioned media of the expanded
hybridomas were then re-screened to identify stable hybridomas
capable of secreting antibodies of interest. Competitive ELISAs
were performed on the 60 highest titer stable hybridomas.
Of the 55 mice and almost 1,500 hybridomas screened, one hybridoma
was discovered that displayed performance characteristics that
justified limited dilution cloning, as is required to ultimately
generate a true monoclonal antibody. This process yielded 47
clones, the majority of which were deemed positive for producing
S1P antibodies. Of these 47 clones, 6 were expanded into 24-well
plates and subsequently screened by competitive ELISA. From the 4
clones that remained positive, one was chosen to initiate
large-scale production of the S1P monoclonal antibody. SCID mice
were injected with these cells and the resulting ascites was
protein A-purified (50% yield) and analyzed for endotoxin levels
(<3 EU/mg). For one round of ascites production, 50 mice were
injected, producing a total of 125 mL of ascites. The antibodies
were isotyped as IgG1 kappa, and were deemed >95% pure by HPLC.
The antibody was prepared in 20 mM sodium phosphate with 150 mM
sodium chloride (pH 7.2) and stored at -70.degree. C. This antibody
is designated LT1002 or Sphingomab.TM..
The positive hybridoma clone (designated as clone 306D326.26) was
deposited with the ATCC (safety deposit storage number SD-5362),
and represents the first murine mAb directed against S1P. The clone
also contains the variable regions of the antibody heavy and light
chains that could be used for the generation of a "humanized"
antibody variant, as well as the sequence information needed to
construct a chimeric antibody.
Screening of serum and cell supernatant for S1P-specific antibodies
was by direct ELISA using a thiolated S1P analog as the antigen. A
standard ELISA was performed, as described below, except that 50 ul
of sample (serum or cell supernatant) was diluted with an equal
volume of PBS/0.1% Tween-20 (PBST) during the primary incubation.
ELISAs were performed in 96-well high binding ELISA plates (Costar)
coated with 0.1 .mu.g of chemically-synthesized thiolated-S1P
conjugated to BSA in binding buffer (33.6 mM Na.sub.2CO.sub.3, 100
mM NaHCO.sub.3; pH 9.5). The thiolated-S1P-BSA was incubated at
37.degree. C. for 1 hr. at 4.degree. C. overnight in the ELISA
plate wells. The plates were then washed four times with PBS (137
mM NaCl, 2.68 mM KCl, 10.14 mM Na.sub.2HPO.sub.4, 1.76 mM
KH.sub.2PO.sub.4; pH 7.4) and blocked with PBST for 1 hr. at room
temperature. For the primary incubation step, 75 uL of the sample
(containing the S1P to be measured), was incubated with 25 uL of
0.1 ug/mL anti-S1P mAb diluted in PBST and added to a well of the
ELISA plate. Each sample was performed in triplicate wells.
Following a 1 hr. incubation at room temperature, the ELISA plates
were washed four times with PBS and incubated with 100 ul per well
of 0.1 ug/mL HRP goat anti-mouse secondary (Jackson Immunoresearch)
for 1 hr. at room temperature. Plates were then washed four times
with PBS and exposed to tetramethylbenzidine (Sigma) for 1-10
minutes. The detection reaction was stopped by the addition of an
equal volume of 1M H.sub.2SO.sub.4. Optical density of the samples
was determined by measurement at 450 nm using an EL-X-800 ELISA
plate reader (Bio-Tech).
For cross reactivity, a competitive ELISA was performed as
described above, except for the following alterations. The primary
incubation consisted of the competitor (S1P, SPH, LPA, etc.) and a
biotin-conjugated anti-S1P mAb. Biotinylation of the purified
monoclonal antibody was performed using the EZ-Link
Sulfo-NHS-Biotinylation kit (Pierce). Biotin incorporation was
determined as per kit protocol and ranged from 7 to 11 biotin
molecules per antibody. The competitor was prepared as follows:
lipid stocks were sonicated and dried under argon before
reconstitution in DPBS/BSA [1 mg/ml fatty acid free BSA
(Calbiochem) in DPBS (Invitrogen 14040-133)]. Purified anti-S1P mAb
was diluted as necessary in PBS/0.5% Triton X-100. Competitor and
antibody solutions were mixed together so to generate 3 parts
competitor to 1 part antibody. A HRP-conjugated streptavidin
secondary antibody (Jackson Immunoresearch) was used to generate
signal.
Another aspect of the competitive ELISA data (shown in FIG. 1,
panel A) is that it shows that the anti-S1P mAb was unable to
distinguish the thiolated-S1P analog from the natural S1P that was
added in the competition experiment. It also demonstrates that the
antibody does not recognize any oxidation products since the analog
was constructed without any double bonds. The anti-S1P mAb was also
tested against natural product containing the double bond that was
allowed to sit at room temperature for 48 hours. Reverse phase HPLC
of the natural S1P was performed according to methods reported
previously (Deutschman, et al. (July 2003), Am Heart J., vol.
146(1):62-8), and the results showed no difference in retention
time. Further, a comparison of the binding characteristics of the
monoclonal antibody to the various lipids shown in FIG. 1, panel A,
indicates that the epitope recognized by the antibody do not
involve the hydrocarbon chain in the region of the double bond of
natural S1P. On the other hand, the epitope recognized by the
monoclonal antibody is the region containing the amino alcohol on
the sphingosine base backbone plus the free phosphate. If the free
phosphate is linked with a choline (as is the case with SPC), then
the binding was somewhat reduced. If the amino group is esterfied
to a fatty acid (as is the case with C1P), no antibody binding was
observed. If the sphingosine amino alcohol backbone was replaced by
a glycerol backbone (as is the case with LPA), there the
S1P-specific monoclonal exhibited no binding. These epitope mapping
data indicate that there is only one epitope on S1P recognized by
the monoclonal antibody, and that this epitope is defined by the
unique polar headgroup of S1P.
In a similar experiment using ELISA measurements, suitable control
materials were evaluated to ensure that this anti-S1P monoclonal
antibody did not recognize either the protein carrier or the
crosslinking agent. For example, the normal crosslinker SMCC was
exchanged for IOA in conjugating the thiolated-S1P to BSA as the
laydown material in the ELISA. When IOA was used, the antibody's
binding characteristics were nearly identical to when
BSA-SMCC-thiolated-S1P was used. Similarly, KLH was exchanged for
BSA as the protein that was complexed with thiolated-S1P as the
laydown material. In this experiment, there was also no significant
difference in the binding characteristics of the antibody.
Binding kinetics: The binding kinetics of S1P to its receptor or
other moieties has, traditionally, been problematic because of the
nature of lipids. Many problems have been associated with the
insolubility of lipids. For BIAcore measurements, these problems
were overcome by directly immobilizing S1P to a BIAcore chip.
Antibody was then flowed over the surface of the chip and
alterations in optical density were measured to determine the
binding characteristics of the antibody to S1P. To circumvent the
bivalent binding nature of antibodies, S1P was coated on the chip
at low densities. Additionally, the chip was coated with various
densities of S1P (7, 20, and 1000 RU) and antibody binding data was
globally fit to a 1:1 interaction model. The results shown in FIG.
2 demonstrate the changes in optical density due to the binding of
the monoclonal antibody to S1P at three different densities of S1P.
Overall, the affinity of the monoclonal antibody to S1P was
determined to be very high, in the range of approximately 88
picomolar (pM) to 99 nM, depending on whether a monovalent or
bivalent binding model was used to analyze the binding data.
Example 2
ELISA Assays
1. Quantitative ELISAs
Microtiter ELISA plates (Costar, Cat No. 3361) were coated with
rabbit anti-mouse IgG, F(ab').sub.2 fragment specific antibody
(Jackson, 315-005-047) diluted in IM Carbonate Buffer (pH 9.5) at
37.degree. C. for 1 h. Plates were washed with PBS and blocked with
PBS/BSA/Tween-20 for 1 hr at 37.degree. C. For the primary
incubation, dilutions of non-specific mouse IgG or human IgG, whole
molecule (used for calibration curve) and samples to be measured
were added to the wells. Plates were washed and incubated with 100
ul per well of HRP conjugated goat anti-mouse (H+L) diluted
1:40,000 (Jackson, cat No 115-035-146) for 1 hr at 37.degree. C.
After washing, the enzymatic reaction was detected with
tetramethylbenzidine (Sigma, cat No T0440) and stopped by adding 1
M H.sub.2SO.sub.4. The optical density (OD) was measured at 450 nm
using a Thermo Multiskan EX. Raw data were transferred to GraphPad
software for analysis.
2. Direct ELISAs
Microtiter ELISA plates (Costar, Cat No. 3361) were coated with
LPA-BSA diluted in 1M Carbonate Buffer (pH 9.5) at 37.degree. C.
for 1 h. Plates were washed with PBS (137 mM NaCl, 2.68 mM KCl,
10.1 mM Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4; pH 7.4) and
blocked with PBS/BSA/Tween-20 for 1 h at room temperature or
overnight at 4.degree. C. The samples to be tested were diluted at
0.4 ug/mL, 0.2 ug/mL, 0.1 ug/mL, 0.05 ug/mL, 0.0125 ug/mL, and 0
ug/mL and 100 ul added to each well. Plates were washed and
incubated with 100 ul per well of HRP conjugated goat anti-mouse
(1:20,000 dilution) (Jackson, cat. no. 115-035-003) for 1 h at room
temperature. After washing, the enzymatic reaction was detected
with tetramethylbenzidine (Sigma, cat. no. T0440) and stopped by
adding 1 M H.sub.2SO.sub.4. The optical density (OD) was measured
at 450 nm using a Thermo Multiskan EX. Raw data were transferred to
GraphPad software for analysis.
3. Competition Assays
The specificity of mAbs was tested in ELISA assays. Microtiter
plates ELISA plates (Costar, Cat No. 3361) were coated with 18:0
LPA-BSA diluted in IM Carbonate Buffer (pH 9.5) at 37.degree. C.
for 1 h. Plates were washed with PBS (137 mM NaCl, 2.68 mM KCl,
10.1 mM Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4; pH 7.4) and
blocked with PBS/BSA/Tween-20 at 37.degree. C. for 1 h or overnight
at room temperature. For the primary incubation 0.4 ug/mL anti-LPA
mAb and designated amounts of (14:0, 16:0, 18:0, 18:1, 18:2 and
20:4) LPA, DSPA, 18:1 LPC (lysophosphatidylcholine), S1P, ceramide
and ceramide-1-phosphate were added to wells of the ELISA plates
and incubated at 37.degree. C. for 1 h. Plates were washed and
incubated with 100 ul per well of HRP conjugated goat anti-mouse
(1:20,000 dilution) (Jackson, cat No 115-035-003) or HRP conjugated
goat anti-human (H+L) diluted 1:50,000 (Jackson, cat No
109-035-003) at 37.degree. C. for 1 h. After washing, the enzymatic
reaction was detected with tetramethylbenzidine and stopped by
adding 1 M H.sub.2SO.sub.4. The optical density (OD) was measured
at 450 nm using a Thermo Multiskan EX. Raw data were transferred to
GraphPad software for analysis.
Example 3
SPHINGOMAB Murine mAb is Highly Specific for S1P
A competitive ELISA demonstrates SPHINGOMAB's specificity for S1P
compared to other bioactive lipids. SPHINGOMAB demonstrated no
cross-reactivity to sphingosine (SPH), the immediate metabolic
precursor of S1P or lysophosphatidic acid (LPA), an important
extracellular signaling molecule that is structurally and
functionally similar to S1P. SPHINGOMAB did not recognize other
structurally similar lipids and metabolites, including
ceramide-1-phosphate (C1P), dihydrosphingosine (DH-SPH),
phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), or
sphingomyelin (SM). SPHINGOMAB did cross react with
dihydrosphingosine-1-phosphate (DH-S1P) and, to a lesser extent,
sphingosylphorylcholine (SPC) (FIG. 3).
Example 4
SPHINGOMAB Significantly Reduces CNV and Scar Formation in a Murine
Model of CNV
Female C57BL6/J mice were subjected to laser-induced rupture of
Bruch's membrane and administered either 0.5 .mu.g of Sphingomab or
an isotype-matched non-specific (NS) antibody diluted in 2 .mu.l of
physiological saline. Mice were sacrificed 14 and 28 days after
laser rupture.
To induce CNV lesions, the pupils were dilated with ophthalmic
tropicamide (0.5%) and phenylephrine (2.5%). A coverslip was placed
on the eye. An Oculight GL 532 nm (Iridex Corporation, Mountain
View, Calif.) coupled to a slit lamp set to deliver a 100 msec
pulse at 150 mW with a 50 .mu.m spot size was used to rupture
Bruch's membrane in three quadrants of the right eye located
approximately 50 .mu.m from the optic disc at relative 9, 12 and 3
o'clock positions. The left eye served as an uninjured control in
all cases. Any lesion not associated with a vapor bubble or lesions
that became confluent were excluded from analysis.
To measure CNV lesion size, choroidal flatmounts of the
sclera-choroid-RPE complex were prepared and stained for
vasculature (R. communis agglutinin I; red) and pericytes (CD140b;
green). Digital images were captured using an epifluorescence Zeiss
Axioplan 2 with RGB Spot high-resolution digital camera and laser
scanning confocal microscope (BioRad MRC 1024, BioRad Corporation,
Temecula, Calif.). For volumetric analysis, a z-series capture was
used and the sum of lesion area throughout the z-series was
multiplied by the z thickness (4 .mu.m) to obtain the lesion
volume.
To assess collagen deposition, the sclera-choroid-RPE complex was
stained with Masson's Trichrome. The sclera-choroid-RPE complex was
embedded in paraffin and then serially sectioned at a thickness of
6 microns. Approximately 30 sections per lesion were evaluated.
Quantitation of the volume of collagen deposition was calculated in
the same manner as described for CNV lesion volume.
Captured digital images were evaluated morphometrically using
ImageJ software (Research Services Branch, National Institutes of
Health, Bethesda, Md.). FIG. 4A shows that SPHINGOMAB dramatically
attenuates choroidal neovascularization 14 and 28 days after
laser-induced rupture of Bruch's membrane. FIG. 4B shows that
SPHINGOMAB significantly reduces fibrosis associated with CNV
lesion formation 28 days after laser-induced rupture of Bruch's
membrane.
Example 5
SPHINGOMAB Inhibits Neovascularization Through Multiple Mechanisms
Including Inhibition of Endothelial Cell Migration and Tube
Formation
S1P promotes the migration of human umbilical vein endothelial
cells (HUVECs) and, in Matrigel and other assays, the formation of
de novo BV formation in vitro; SPHINGOMAB can neutralize these
effects of S1P. Experiments were performed as described by Visentin
et al. (Cancer Cell 2006 March; 9(3):225-38). Data in FIG. 5A
suggest that HUVECs seeded onto GF-reduced Matrigel formed multiple
capillary-like structures in the presence of S1P and failed to form
capillary-like structures in the absence of S1P or when
co-incubated with SPHINGOMAB and S1P. Data in FIG. 5B demonstrate
the potent ability of 0.1-1 .mu.M S1P to stimulate HUVEC migration
2-2.5 fold over non-treated HUVECs, or HUVECs co-incubated with
SPHINGOMAB in a Matrigel chemoinvasion assay. Combined, these
studies demonstrate that SPHINGOMAB can efficiently mitigate the
pro-angiogenic effects of S1P on ECs.
Example 6
SPHINGOMAB Inhibits Neovascularization Through Multiple Mechanisms
Including Mitigation of the Effects of S1P, VEGF and bFGF In
Vivo
Based on in vivo studies showing that S1P increased endothelial
capillary growth into subcutaneously implanted Matrigel plugs, we
speculated that SPHINGOMAB could reduce de novo BV formation in
vivo. To investigate this, we employed the in vivo Matrigel Plug
assay for neovascularization. In one set of experiments, Matrigel
was supplemented with either 1 .mu.M S1P, 0.5 .mu.g/mL bFGF or 1
.mu.g/mL VEGF and then injected I.P. into mice (n=4). After 10
days, the mice were heparinized and injected with the fluorescent
lectin, Isolectin B4-FITC, which binds to adhesion molecules
expressed by vascular EC that form the growing BVs. The plugs were
then excised, frozen in OCT, sectioned and viewed for FITC-stained
BVs. Data in FIG. 6A suggest that S1P is a more potent stimulator
of neovascularization in vivo than bFGF or VEGF [Lee, et al.,
(1999), Biochem Biophys Res Commun., vol 264: 743-50], as evidenced
by the vast amount of FITC-stained BVs in the plugs containing S1P
compared to the plugs containing bFGF or VEGF.
Sections of the plugs were then stained with hemotoxyln & eosin
for evaluation of EC infiltration (FIG. 6B). The infiltration of
ECs is a critical step in neo-vascularization. Plugs containing S1P
had a 3-fold increase of EC infiltration in comparison to the
Matrigel only plugs. Cell infiltration is presumed to be ECs
although we recognize that other cell types such as immune cells
may also be stained. Mice systemically administered SPHINGOMAB
every 48 hrs (initiated 1 day prior to plug implantation),
demonstrated a reduced amount of EC infiltration even when S1P was
added to the Matrigel plugs. These results demonstrate the ability
of SPHINGOMAB to inhibit EC infiltration in vivo.
Endogenous S1P from the blood and surrounding tissue could supply a
wound with pro-angiogenic stimuli. The ability of SPHINGOMAB to
reduce endogenous S1P in a wound was investigated. Optimally
stimulated plugs (Matrigel supplemented with 0.5 .mu.g/mL bFGF or
10 mg/mL VEGF) were implanted into mice. Mice received i.p.
injections of 25 mg/kg SPHINGOMAB or saline every 48 hrs starting 1
day prior to Matrigel implantation. Each treatment group (Matrigel,
Matrigel plus GF or Matrigel plus GF and administered SPHINGOMAB)
consisted of a minimum of 6 mice. After 10 days, the mice were
treated with heparin, injected with Isolectin B4-FITC, the plugs
excised, embedded in OCT freezing medium and sectioned.
Micro-vascular density was qualitatively accessed by lectin-FITC
stained vessels as shown in FIG. 6C. BV staining was sporadic in
control (untreated) plugs, whereas the plugs containing bFGF or
VEGF demonstrated significant evidence of vascularization. The
plugs from mice treated with the SPHINGOMAB demonstrated a
significant reduction in BV formation compared to the bFGF or VEGF
plugs from saline-treated mice. Quantification of stained vessels
revealed a 5 to 8.5-fold decrease in neovascularization of VEGF- or
bFGF-containing plugs, respectively, from animals treated with
SPHINGOMAB in comparison to saline-treated animals (FIG. 6C). This
evaluation further demonstrates the ability of endogenous serum and
tissue S1P to enhance micro-vascularization as well as the ability
of SPHINGOMAB to neutralize endogenous S1P's pro-angiogenic
effects.
Example 7
SPHINGOMAB Inhibits Scar Formation In Vivo
S1P makes profound contributions to wound healing by activating
fibroblast migration, proliferation and collagen production;
SPHINGOMAB neutralizes these effects. Several studies using
multiple types of fibroblasts confirm S1P's ability to promote
wound healing: 1) S1P increased Swiss-3T3 fibroblast proliferation
as measured by .sup.3H-thymidine incorporation using standard
methods (FIG. 7A); 2) S1P promoted the migration of cardiac
fibroblasts in a standard scratch wound healing assay. (FIG. 7B);
3) S1P promoted collagen expression by cardiac fibroblasts isolated
from transgenic mice possessing the collagen 1a GFP reporter, as
indicated by immunofluorescence microscopy (FIG. 7C); and 4) S1P
induced the differentiation of WI-38 lung fibroblasts into
myofibroblasts, cells that are active in scar remodeling, as
indicated by increased expression of myofibroblast marker protein,
smooth muscle actin, using immunoblot analysis (FIG. 7D). In each
of these assays, SPHINGOMAB neutralized S1P's. It is anticipated
that ocular fibroblasts would respond similarly to S1P and
SPHINGOMAB. Similarities between cardiovascular disease and
neovascular lesions of AMD, including scar remodeling and
subsequent, maladaptive fibrous tissue formation, have been noted
(Vine, et al. (2005), Opthalmology., vol 112: 2076-80 and Seddon
and Chen (2004), Int Opthalmol Clin., vol 44: 17-39); thus, it is
believed that SPHINGOMAB would have effects on ocular
neovascularization and scarring similar to those it has
demonstrated in cardiovascular systems.
Studies evaluated the efficacy of SPHINGOMAB to reduce cardiac scar
formation after permanent myocardial infarction (MI) via ligation
of the left descending coronary artery in mice. Systemic
administration of 25 mg/kg SPHINGOMAB or saline was initiated 48
hrs after surgery. Antibody administration at 48 hr. was chosen to
allow normal, reparative scar formation to occur during the early
remodeling phase and permit beneficial, S1P-stimulated angiogenesis
immediately after the MI. Two weeks after the infarct, mice were
sacrificed and fibrosis was accessed by Masson's trichrome staining
of the cardiac tissue. Animals receiving SPHINGOMAB treatments
exhibited almost complete abrogation of perivascular fibrosis (FIG.
7, photos). As a control for any non-specific wound-healing
responses, sham animals underwent thoracotomy without coronary
artery ligation (FIG. 7E).
Example 8
S1P Promotes Transformation of Ocular Epithelial Cells and
Fibroblasts into Contractile, Scar Tissue-producing
Myofibroblasts
Pathological tissue fibrosis (scar formation) is a primary,
contributing factor in a number of ocular disorders, including:
age-related macular degeneration, diabetic retinopathy, retinopathy
of prematurity, proliferative vitreoretinopathy and consequences of
glaucoma surgery.
In many of these disorders, circulating growth factors and
chemokines promote the transformation of normal ocular cells into
fibrocontractile, scar tissue-producing cells that have been termed
"myofibroblasts". Normally, myofibroblasts are responsible for
tissue repair as part of the wound healing response following
injury. However altered number and function of myofibroblasts are
implicated in diseases characterized by pathological scar tissue
formation in the liver, skin, lung, kidney, heart and eyes. In the
eye, transformation of retinal pigmented epithelial (RPE) cells to
a myofibroblast phenotype is linked to formation of
fibro-contractile membranes which cause retinal detachment and
subsequent vision impairment. In addition, myofibroblast
transformation of ocular fibroblasts can result in abnormal scar
tissue production after eye injury leading to subsequent vision
loss. Although many of the circulating protein factors in the eye
that promote myofibroblast formation have been identified, nothing
is known regarding the role of lysolipids such as S1P in this
process. Therefore, we examined the effects of S1P on myofibroblast
transformation of several human ocular cell lines. As shown in FIG.
8, S1P stimulates production of .alpha.-Smooth muscle actin
(.alpha.-SMA; a myofibroblast marker) in human retinal pigmented
epithelial cells (FIG. 8A) and human conjunctiva fibroblasts (FIG.
8B). These data demonstrate for the first time, that S1P is among
the milieu of circulating chemical factors that promote
transformation of ocular epithelial cells and fibroblasts into
contractile, scar tissue-producing myofibroblasts which may
contribute to retinal detachment, ocular fibrosis and subsequent
vision impairment.
In these experiments, the ability of S1P to promote .alpha.-SMA
expression differed in a concentration dependent manner between the
retinal pigmented epithelial cells and conjunctiva fibroblasts. As
shown, a significant increase in .alpha.-SMA expression was
observed at the 0.001 .mu.M concentration in the epithelial cells
which then decreased to basal levels at the 10 .mu.M concentration.
In contrast, a significant increase in .alpha.-SMA expression was
observed only at the 10 .mu.M concentration in the conjunctiva
fibroblasts. This difference is believed to result from increased
S1P receptor expression in the epithelial cells compared to the
fibroblasts. Due to increased S1P receptor expression levels,
retinal pigmented epithelial cells are likely more sensitive to S1P
at low concentrations. In contrast, at high S1P levels the
receptors become sensitized or possibly even internalized leading
to decreased stimulation by S1P.
Collagen is one of the primary structural proteins that supports
all tissues in the body and is one of the main components of scar
tissue. In the non-pathological setting, total collagen content
within tissue is maintained via a balance between collagen
production by fibroblasts and degradation by certain enzymes. A
number of disorders that involve increased levels of scar tissue
result, in part, from physiological and molecular processes that
inhibit degradation of collagen that is need for scar formation. It
was hypothesized that the ability of S1P to promote scar tissue
formation may result from its ability to inhibit collagen
degradation, thereby leading to net increases in scar tissue within
organs. Therefore, the effects of S1P on expression of plasminogen
activator inhibitor (PAI-1) in human conjunctiva fibroblasts were
examined. Increased PAI-1 expression correlates with a decrease in
the proteolytic degradation of connective tissue and is upregulated
in association with several fibrotic diseases that involve
increased scarring. As shown in FIG. 8C, S1P stimulates the PAI-1
expression in a dose-dependent manner. These data suggest that, may
also promote scar tissue formation by stimulating the expression of
proteins that inhibit its degradation, suggesting that S1P
functions through multiple mechanistic pathways to promote and
maintain pathological scarring associated with ocular diseases.
Example 9
SPHINGOMAB Inhibits Inflammatory and Immune Cell Infiltration
Inflammation is the first response in the remodeling process. It is
triggered both by ischemia and by cellular damage and results in
up-regulation of cytokine expression which stimulates the migration
of macrophages and neutrophils to the injured area for phagocytosis
of dead cells and to further up-regulate the inflammatory response
[Jordan, et al. (1999), Cardiovasc Res., vol 43: 860-78]. Mast
cells are also important cellular mediators of the inflammatory
response. S1P released from mast cells is responsible for many of
the adverse responses seen in experimental animal models of
inflammation [Jolly, et al (2004), J Exp Med., vol 199: 959-70 and
Jolly et al (2005), Blood., vol 105: 4736-42].
Based upon the similarities of immune and inflammatory responses in
CNV and CVD, the efficacy of SPHINGOMAB to mitigate immune cell
infiltration into a wound was evaluated in a murine infarct model
as an indication of SPHINGOMAB's potential effects in mitigating
these damages during AMD [Vine, et al. (2005), Opthalmology., vol
112: 2076-80; and Seddon and Chen (2004), Int Opthalmol Clin., vol
44: 17-39]. Four days post-MI, macrophage and mast cell
infiltration was evaluated using MAC-1 and MCG35 antibodies,
respectively, within the area at risk. SPHINGOMAB dramatically
attenuated the density of inflammatory macrophages (FIG. 9A) and
mast cells (FIG. 9B) suggesting that SPHINGOMAB may neutralize
immune and inflammatory damages during AMD.
Example 10
Cloning and Characterization of the Variable Domains of an S1P
Murine Monoclonal Antibody (LT1002; Sphingomab)
This example reports the cloning of the murine mAb against S1P. The
overall strategy consisted of cloning the murine variable domains
of both the light chain (VL) and the heavy chain (VH). The
consensus sequence of 306D VH shows that the constant region
fragment is consistent with a gamma 2b isotype. The murine variable
domains were cloned together with the constant domain of the light
chain (CL) and with the constant domain of the heavy chain (CH1,
CH2, and CH3), resulting in a chimeric antibody construct.
1. Cloning of the murine mAb
A clone from the anti-S1P hybridoma cell line 306D326.1
(ATCC#SD-5362) was grown in DMEM (Dulbecco's Dulbecco's Modified
Eagle Medium with GlutaMAX.TM. I, 4500 mg/L D-Glucose, Sodium
Puruvate; Gibco/Invitrogen, Carlsbad, Calif., 111-035-003), 10% FBS
(Sterile Fetal Clone I, Perbio Science), and 1.times.
glutamine/Penicillin/Streptomycin (Gibco/Invitrogen). Total RNA was
isolated from 10.sup.7 hybridoma cells using a procedure based on
the RNeasy Mini kit (Qiagen, Hilden Germany). The RNA was used to
generate first strand cDNA following the manufacturer's protocol
(1.sup.st strand synthesis kit, Amersham Biosciences).
The immunoglobulin heavy chain variable region (VH) cDNA was
amplified by PCR using an MHV7 primer (MHV7:
5'-ATGGRATGGAGCKGGRTCTTTMTCTT-3' [SEQ ID NO: 1]) in combination
with a IgG2b constant region primer MHCG1/2a/2b/3 mixture (MHCG1:
5'-CAGTGGATAGACAGATGGGGG-3' [SEQ ID NO: 2]; MHCG2a:
5'-CAGTGGATAGACCGATGGGGC-3 [SEQ ID NO: 3]; MHCG2b:
5'-CAGTGGATAGACTGATGGGGG-3' [SEQ ID NO: 4]; MHCG3:
5'-CAAGGGATAGACAGATGGGGC-3' [SEQ ID NO: 5]). The product of the
reaction was ligated into the pCR2.1.RTM.-TOPO.RTM. vector
(Invitrogen) using the TOPO-TA cloning.RTM. kit and sequence. The
variable domain of the heavy chain was then amplified by PCR from
this vector and inserted as a Hind III and Apa I fragment and
ligated into the expression vector pG1D200 (see U.S. Pat. No.
7,060,808) or pG4D200 (id.) containing the HCMVi promoter, a leader
sequence, and the gamma-1 constant region to generate the plasmid
pG1D200306DVH (FIG. 10). The consensus sequence of 306D V.sub.H
(shown below) showed that the constant region fragment was
consistent with a gamma 2b isotype.
Similarly, the immunoglobulin kappa chain variable region (VK) was
amplified using the MKV 20 primer (5'-GTCTCTGATTCTAGGGCA-3' [SEQ ID
NO: 6]) in combination with the kappa constant region primer MKC
(5'-ACTGGATGGTGGGAAGATGG-3' [SEQ ID NO: 7]). The product of this
reaction was ligated into the pCR2.1.RTM.-TOPO.RTM. vector using
the TOPO-TA cloning.RTM. kit and sequence. The variable domain of
the light chain was then amplified by PCR and then inserted as a
Bam HI and Hind III fragment into the expression vector pKN100 (see
U.S. Pat. No. 7,060,808) containing the HCMV promoter, a leader
sequence, and the human kappa constant domain, generating plasmid
pKN100306DVK.
The heavy and light chain plasmids pG1D200306DVH plus pKN100306DVK
were transformed into DH4a bacteria and stocked in glycerol.
Large-scale plasmid DNA was prepared as described by the
manufacturer (Qiagen, endotoxin-free MAXIPREP.TM. kit). DNA
samples, purified using Qiagen's QIAprep Spin Miniprep Kit or
EndoFree Plasmid Mega/Maxi Kit, were sequenced using an ABI 3730xl
automated sequencer, which also translates the fluorescent signals
into their corresponding nucleobase sequence. Primers were designed
at the 5' and 3' ends so that the sequence obtained would overlap.
The length of the primers was 18-24 bases, and preferably they
contained 50% GC content and no predicted dimers or secondary
structure. The amino acid sequences for the mouse V.sub.H and
V.sub.L domains from Sphingomab.TM. are SEQ ID NOS: 8 and 9,
respectively (Table 2). The CDR residues (see Kabat, E A (1982),
Pharmacol Rev, vol. 34: 23-38) are underlined in Table 2, and are
shown separately below in Table 3.
TABLE-US-00002 TABLE 2 V.sub.H and V.sub.L domains from the murine
mAb, Sphingomab.TM. mouse
QAHLQQSDAELVKPGASVKISCKVSGFIFIDHTIHWMKQRPEQG SEQ ID V.sub.H
LEWIGCISPRHDITKYNEMFRGKATLTADKSSTTAYIQVNSLTF NO: 8 domains
EDSAVYFCARGGFYGSTIWfDFWGQGTTLTVS mouse V.sub.L
ETTVTQSPASLSMAIGEKVTIRCITTTDIDDDMNWFQQKPGEPPNLLISE SEQ ID domains
GNILRPGVPSRFSSSGYGTDFLFTIENMLSEDVADYYCLQSDNLPFTFGS NO: 9
GTKLEIK
TABLE-US-00003 TABLE 3 Mouse Sphingomab.TM. CDR sequences of the
mouse V.sub.H and V.sub.L domains CDR V.sub.L CDR ITTTDIDDDMN (SEQ
ID NO: 10) CDR1 EGNILRP (SEQ ID NO: 11) CDR2 LQSDNLPFT (SEQ ID NO:
12) CDR3 V.sub.H CDR DHTIH (SEQ ID NO: 13) CDR1 CISPRHDITKYNEMFRG
(SEQ ID NO: 14) CDR2 GGFYGSTIWFDF (SEQ ID NO: 15) CDR3
The amino acid sequences of several chimeric antibody variable
(V.sub.H and V.sub.L) domains are compared in Table 4. These
variants were cloned in the Lonza expression vectors. Sequences of
the murine V.sub.H and V.sub.L domains were used to construct a
molecular model to determine which framework residues should be
incorporated into the humanized antibody.
TABLE-US-00004 TABLE 4 Amino acid sequences of the humanized
V.sub.H and V.sub.L domains from the humanized anti-S1P antibody
variants V.sub.H Variants pATH200
mgstailalllavlqgvcsevqlvqsgaevkkpgeslkiscqsfgyifidhtihwvrqmpgqglew-
mgcisprhditkyn SEQ ID NO: 16 pATH201
.......................................................m..........-
.............. SEQ ID NO: 39 pATH202
.............................................f.........m..........-
i............. SEQ ID NO: 40 pATH203
..................................................................-
i............. SEQ ID NO: 41 pATH204
.............................................f....................-
.............. SEQ ID NO: 42 pATH205
.............................................f.........m..........-
i............. SEQ ID NO: 43 pATH206
....................a........................f.........m..........-
i............. SEQ ID NO: 44 pATH207
.......................................................m..........-
..a........... SEQ ID NO: 45 Sequences Continue: pATH200
emfrgqvtisadkssstaylqwsslkasdtamyfcarggfygstiwfdfwgqgtmvtvssastkgp-
s continued pATH201
..................................................................-
. pATH202
..................................................................-
. pATH203
..................................................................-
. pATH204
..................................................................-
. pATH205
......a.l.........................................................-
. pATH206
......a.l.........................................................-
. pATH207
..................................................................-
. V.sub.L Variants pATH300
mdmrvpaqllgllllwlpgarcettltqspsflsasvgdrvtitcitttdidddmnwyqqepgkap-
klliyegnilrpgv (SEQ ID NO: 17) pATH301
..................................................................-
....s......... SEQ ID NO: 46 pATH302
.........................................................f........-
.............. SEQ ID NO: 47 pATH303
.........................v........................................-
....s......... SEQ ID NO: 48 pATH304
.........................................................f........-
....s......... SEQ ID NO: 49 pATH305
.........................v...............................f........-
....s......... SEQ ID NO: 50 pATH306
.........................v...............................f........-
....s......... SEQ ID NO: 51 pATH308
.........................v...............................f........-
....s......... SEQ ID NO: 52 pATH309
..................................................................-
....s......... SEQ ID NO: 53 Sequences continue pATH300
psrfsgsgsgtdftltisklqpedfatyyclqsdnlpftfgqgtkleikrewip continued
pATH301 ......................................................
pATH302 ......................................................
pATH303 .....................................................-
pATH304 ......................................................
pATH305 ....................................................--
pATH306 .....s..............................................--
pATH308 .....s..y.............................................
pATH309 .....s..y.............................................
Corresponding nucleotide sequences are shown in Table 5:
TABLE-US-00005 TABLE 5 pATH and CDR sequences SEQ ID Name Sequence
NO: CDR1 V.sub.L: ataaccaccactgatattgatgatgatatgaac 18 CDR2
V.sub.L: gaaggcaatattcttcgtcct 19 CDR3 V.sub.L:
ttgcagagtgataacttaccattcacg 20 CDR1 V.sub.H gaccatacttcac 21 CDR2
V.sub.H: tgtatttctcccagacatgatattactaaatacaatgagatgttcaggggc 22
CDR3 V.sub.H: ggggggttctacggtagtactatctggtttgacttt 23 CDR2 V.sub.H
gctatttctcccagacatgatattactaaatacaatgagatgttcaggggc 24 (pATH 207):
pATH200 cgccaagcttgccgccaccatggggtcaaccgccatcctcgccctcctcctg 25
nucleotide gctgttctccaaggagtctgttccgaggtgcagctggtgcagtctggagcag
sequence: aggtgaaaaagcccggggagtctctgaagatctcctgtcagagttttggatac
atctttatcgaccatacttcactgggtgcgccagatgcccgggcaaggcctg
gagtggatgtgtatttctcccagacatgatattactaaatacaatgagatgttca
ggggccaggtcaccatctcagccgacaagtccagcagcaccgcctacttgc
agtggagcagcctgaaggcctcggacaccgccatgtatttctgtgcgagag
gggggttctacggtagtactatctggtttgacttttggggccaagggacaatg
gtcaccgtctcttcagcctccaccaagggcccatcg pATH207
cgccaagcttgccgccaccatggggtcaaccgccatcctcgccctcctcctg 26 nucleotide
gctgttctccaaggagtctgttccgaggtgcagctggtgcagtctggagcag sequence:
aggtgaaaaagcccggggagtctctgaagatctcctgtcagagttttggatac
atcgaccatacttcactggatgcgccagatgcccgggcaaggcctggagtg
gatgggggctatttctcccagacatgatattactaaatacaatgagatgttcag
gggccaggtcaccatctcagccgacaagtccagcagcaccgcctacttgca
gtggagcagcctgaaggcctcggacaccgccatgtatttctgtgcgagagg
ggggttctacggtagtactatctggtttgacttttggggccaagggacaatggt
caccgtctcttcagcctccaccaagggcccatcg pATH207
mgstailalllavlqgvcsevqlvqsgaevkkpgeslkiscqsfgyifidhti 27 amino acid
hwmrqmpgqglewmgaisprhditkynemfrgqvtisadkssstaylq sequence
wsslkasdtamyfcarggfygstiwfdfwgqgtmvtvssastkgps pATH300
cgccaagcttgccgccaccatggacatgagggtccccgctcagctcctggg 28 nucleotide
gctcctgctgctctggctcccaggtgccagatgtgaaacgacactcacgcag sequence:
tctccatccttcctgtctgcatctgtaggagacagagtcaccatcacataacca
ccactgatattgatgatgatatgaactggtatcagcaggaaccagggaaagc
ccctaagctcctgatctatgaaggcaatattcttcgtcctggggtcccatcaag
gttcagcggcagtggatctggcacagatttcactctcaccatcagcaaattgc
agcctgaagattttgcaacttattactgtttgcagagtgataacttaccattcacg
ttcggccaagggaccaagctggagatcaaacgtgagtggatcccgcg pATH308
cgccaagcttgccgccaccatggacatgagggtccccgctcagctcctggg 29 nucleotide
gctcctgctgctctggctcccaggggccagatgtgaaacgacagtgacgca sequence
gtctccatccttcctgtctgcatctgtaggagacagagtcaccatcacttgcata
accaccactgatattgatgatgatatgaactggttccagcaggaaccaggga
aagcccctaagctcctgatctccgaaggcaatattcttcgtcctggggtcccat
caagattcagcagcagtggatatggcacagatttcactctcaccatcagcaaa
ttgcagcctgaagattttgcaacttattactgtttgcagagtgataacttaccatt
cactttcggccaagggaccaagctggagatcaaac pATH308
mrvpaqllgllllwlpgarcettvtqspsflsasvgdrvtitcitttdidddmn 30 amino
acid wfqepgkapkllisegnilrpgvpsrfsssgygtdftltisklqpedfatyycl
sequence qsdnlpftfgqgtkleik
2. Expression and Binding Properties of the Chimeric Antibody
The heavy and light chain plasmids of both pG1D200306DVH plus
pKN100306DVK were transformed into DH4a bacteria and stocked in
glycerol. Large scale plasmid DNA was prepared as described by the
manufacturer (Qiagen, endotoxin-free MAXIPREP.TM. kit Cat. No.
12362).
For antibody expression in a non-human mammalian system, plasmids
were transfected into the African green monkey kidney fibroblast
cell line COS 7 by electroporation (0.7 ml at 10.sup.7 cells/ml)
using 10 ug of each plasmid. Transfected cells were plated in 8 ml
of growth medium for 4 days. The chimeric 306DH1.times.306DVK-2
antibody was expressed at 1.5 .mu.g/ml in transiently
co-transfected COS cell conditioned medium. The binding of this
antibody to S1P was measured using the S1P ELISA.
The expression level of the chimeric antibody was determined in a
quantitative ELISA as follows. Microtiter plates (Nunc MaxiSorp
immunoplate, Invitrogen) were coated with 100 .mu.l aliquots of 0.4
.mu.g/ml goat anti-human IgG antibody (Sigma, St. Louis, Mo.)
diluted in PBS and incubate overnight at 4.degree. C. The plates
were then washed three times with 200 .mu.l/well of washing buffer
(1.times.PBS, 0.1% TWEEN). Aliquots of 200 .mu.L of each diluted
serum sample or fusion supernatant were transferred to the
toxin-coated plates and incubated for 37.degree. C. for 1 hr.
Following 6 washes with washing buffer, the goat anti-human kappa
light chain peroxidase conjugate (Jackson Immuno Research) was
added to each well at a 1:5000 dilution. The reaction was carried
out for 1 hr at room temperature, plates were washed 6 times with
the washing buffer, and 150 .mu.L of the K-BLUE substrate (Sigma)
was added to each well, incubated in the dark at room temperature
for 10 min. The reaction was stopped by adding 50 .mu.l of RED STOP
solution (SkyBio Ltd.) and the absorption was determined at 655 nm
using a Microplater Reader 3550 (Bio-Rad Laboratories Ltd.).
Results from the antibody binding assays are shown in FIG. 11.
3. 293F Expression
The heavy and light chain plasmids were transformed into Top 10 E.
coli (One Shot Top 10 chemically competent E. coli cells
(Invitrogen, C4040-10)) and stocked in glycerol. Large scale
plasmid DNA was prepared as described by the manufacturer (Qiagen,
endotoxin-free MAXIPREP.TM. kit CatNo 12362).
For antibody expression in a human system, plasmids were
transfected into the human embryonic kidney cell line 293F
(Invitrogen) using 293fectin (Invitrogen) and using 293F-FreeStyle
Media (Invitrogen) for culture. Light and heavy chain plasmids were
both transfected at 0.5 g/mL. Transfections were performed at a
cell density of 10.sup.6 cells/mL. Supernatants were collected by
centrifugation at 1100 rpm for 5 minutes at 25.degree. C. 3 days
after transfection. Expression levels were quantified by
quantitative ELISA (see previous examples) and varied from
.about.0.25-0.5 g/mL for the chimeric antibody.
4. Antibody Purification
Monoclonal antibodies were purified from culture supernatants by
passing culture supernatants over protein A/G columns (Pierce, Cat.
No 53133) at 0.5 mL/min. Mobile phases consisted of 1.times. Pierce
IgG binding Buffer (Cat. No 21001) and 0.1 M glycine pH 2.7
(Pierce, Elution Buffer, Cat. No 21004). Antibody collections in
0.1 M glycine were diluted 10% (v/v) with 1 M Phosphate Buffer, pH
8.0, to neutralize the pH. IgG, collections were pooled and
dialyzed exhaustively against 1.times.PBS (Pierce Slide-A-Lyzer
Cassette, 3,500 MWCO, Cat. No 66382). Eluates were concentrated
using Centricon YM-3 (10,000 MWCO Amicon Cat. No 4203) by
centrifugation for 1 h at 2,500 rcf. The antibody concentration was
determined by quantitative ELISA as described above using a
commercial myeloma IgG.sub.1 stock solution as a standard. Heavy
chain types of mAbs were determined by ELISA using Monoclonal
Antibody Isotyping Kit (Sigma, ISO-2).
5. Comparative Binding of Antibody Variants to S1P
Table 6, below, shows a comparative analysis of mutants with the
chimeric antibody. To generate these results, bound antibody was
detected by a second antibody, specific for the mouse or human IgG,
conjugated with HRP. The chromogenic reaction was measured and
reported as optical density (OD). The concentration of the panel of
antibodies was 0.1 ug/ml. No interaction of the second antibody
with S1P-coated matrix alone was detected.
TABLE-US-00006 TABLE 6 Comparative binding to S1P on variants of
the chimeric anti-S1P antibody. Variable Domain Mutation Plasmids
Binding Chimeric pATH50 + pATH10 1.5 HC CysAla pATH50 + pATH11 2
CysSer pATH50 + pATH12 0.6 CysArg pATH50 + pATH14 0.4 CysPhe pATH50
+ pATH16 2 LC MetLeu pATH53 + pATH10 1.6
6. Determination of Binding Kinetics by Surface Plasmon Resonance
(SPR)
All binding data were collected on a Biacore 2000 optical biosensor
(Biacore AB, Uppsala Sweden). S1P was coupled to a maleimide CM5
sensor chip. First the CM5 chip was activated with an equal mixture
of NHS/EDC for seven minutes followed by a 7 minute blocking step
with ethyldiamine. Next sulfo-MBS (Pierce Co.) was passed over the
surfaces at a concentration of 0.5 mM in HBS running buffer (10 mM
HEPES, 150 mM NaCl, 0.005% p20, pH 7.4). S1P was diluted into the
HBS running buffer to a concentration of 0.1 mM and injected for
different lengths of time producing 2 different density S1P
surfaces (305 and 470 RU). Next, binding data for the mAb was
collected using a 3-fold dilution series starting with 16.7 nM,
50.0 nM, 50.0 nM, 16.7 nM, and 16.7 nM for the mouse, 201308,
201309, and 207308 antibodies respectively.
Each concentration was tested in duplicate. Surfaces were
regenerated with 50 mM NaOH. All data were collected at 25.degree.
C. Responses data were processed using a reference surface as well
as blank injections. The data sets (responses from two surfaces and
each variant tested twice were fit to interaction models to obtain
binding parameters. Data from the different mAb concentrations were
globally fitted using a 1:1 (mouse) or 1:2 (variants) interaction
model to determine apparent binding rate constants. The number in
parentheses indicates the error in the last digit.
Example 11
Chimeric mAb to S1P
As used herein, the term "chimeric" antibody (or "immunoglobulin")
refers to a molecule comprising a heavy and/or light chain which is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(Cabilly, et al., supra; Morrison et al., Proc. Natl. Acad. Sci.
U.S.A. 81:6851 (1984)).
A chimeric antibody to S1P was generated using the variable regions
(Fv) containing the active S1P binding regions of the murine
antibody from a particular hybridoma (ATCC safety deposit storage
number SD-5362) with the Fc region of a human IgG1 immunoglobulin.
The Fc regions contained the CL, ChL, and Ch3 domains of the human
antibody. Without being limited to a particular method, chimeric
antibodies could also have been generated from Fc regions of human
IgG1, IgG2, IgG3, IgG4, IgA, or IgM. As those in the art will
appreciate, "humanized" antibodies can been generated by grafting
the complementarity determining regions (CDRs, e.g. CDR1-4) of the
murine anti-S1P mAb with a human antibody framework regions (e.g.,
Fr1, Fr4, etc.) such as the framework regions of an IgG1. FIG. 11
shows the binding of the chimeric and full murine mAbs in a direct
ELISA measurement using thiolated-S1P as lay down material.
For the direct ELISA experiments shown in FIG. 11, the chimeric
antibody to S1P had similar binding characteristics to the fully
murine monoclonal antibody. ELISAs were performed in 96-well
high-binding ELISA plates (Costar) coated with 0.1 ug of
chemically-synthesized, thiolated S1P conjugated to BSA in binding
buffer (33.6 mM Na.sub.2CO.sub.3, 100 mM NaHCO.sub.3; pH 9.5). The
thiolated S1P-BSA was incubated at 37.degree. C. for 1 hr. or at
4.degree. C. overnight in the ELISA plate. Plates were then washed
four times with PBS (137 mM NaCl, 2.68 mM KCl, 10.14 mM
Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4; pH 7.4) and blocked
with PBST for 1 hr. at room temperature. For the primary incubation
step, 75 uL of the sample (containing the S1P to be measured), was
incubated with 25 .mu.L of 0.1 .mu.g/mL anti-S1P monoclonal
antibody diluted in PBST and added to a well of the ELISA plate.
Each sample was performed in triplicate wells. Following a 1 hr.
incubation at room temperature, the ELISA plates were washed four
times with PBS and incubated with 100 ul per well of 0.1 ug/mL HRP
goat anti-mouse secondary (Jackson Immunoresearch) for 1 hr. at
room temperature. Plates were then washed four times with PBS and
exposed to tetramethylbenzidine (Sigma) for 1-10 minutes. The
detection reaction was stopped by the addition of an equal volume
of 1 M H.sub.2SO.sub.4. Optical density of the samples was
determined by measurement at 450 nm using an EL-X-800 ELISA plate
reader (Bio-Tech).
Again, the preferred method of measuring either antibody titer in
the serum of an immunized animal or in cell-conditioned media (for
example, supernatant) of an antibody-producing cell such as a
hybridoma, involves coating the ELISA plate with a target ligand
(e.g., a thiolated analog of S1P, LPA, etc.) that has been
covalently linked to a protein carrier such as BSA.
Without being limited to particular method or example, chimeric
antibodies could be generated against other lipid targets such as
LPA, PAF, ceramides, sulfatides, cerebrosides, cardiolipins,
phosphotidylserines, phosphotidylinositols, phosphatidic acids,
phosphotidylcholines, phosphatidylethanolamines, eicosinoids, and
other leukotrienes, etc. Further, many of these lipids could also
be glycosylated and/or acetylated, if desired.
Example 12
Generation and Characterization of Humanized Anti-S1P Monoclonal
Antibody LT1009 (Sonepcizumab)
The murine anti-S1P monoclonal antibody 306D (LT1002;
Sphingomab.TM.), which specifically binds S1P, has been shown to
potently suppress angiogenesis and tumor growth in various animal
models. As discussed below, LT1002 was humanized using sequence
identity and homology searches for human frameworks into which to
graft the murine CDRs and a computer-generated model to guide some
framework backmutations. Two variants, HuMAbHCLC.sub.3 (LT1004)
(with 3 backmutations in the light chain) and HuMAbHCLC.sub.5
(LT1006) (with 5 backmutations in the light chain) exhibited
binding affinity in the nanomolar range. Further engineering was
performed in an effort to improve the biophysical and biological
properties of the humanized variants. The humanized variants
HuMAbHC.sub.CysAlaLC.sub.3 (LT1007) and HuMAbHC.sub.CysAlaLC.sub.5
(LT1009) in which a free-cysteine residue in HCDR2 was replaced
with alanine exhibited a binding affinity in the picomolar range.
All humanized variants inhibited angiogenesis in the choroid
neovascularization (CNV) model of age-related macular degeneration
(AMD), with HuMAbHC.sub.CysAlaLC.sub.5 (LT1009) exhibiting superior
stability and in vivo efficacy compared to the parent murine
antibody. The variant huMAbHC.sub.CysalaLC.sub.5 (LT1009) was
designated Sonepcizumab.TM..
a. Humanization Design for the Anti-S1P Antibody
The variable domains of murine mAb LT1002 (Sphingomab.TM.) were
humanized via CDR grafting (Winter U.S. Pat. No. 5,225,539). The
CDR residues were identified based on sequence hypervariability as
described by Kabat et al. 1991.
In this study, suitable acceptor structures were selected based on
a homology search of human antibodies in the IMGT and Kabat
databases using a structural alignment program (SR v7.6). The
initial step was to query these human heavy variable (VH) and light
variable (VL) sequence databases with LT1002 VH and VL protein
sequences respectively, to identify human frameworks (FR) with high
sequence identity in the FR, at Vernier (Foote, J. & Winter, G.
Antibody framework residues affecting the conformation of the
hypervariable loops. J Mol. Biol. 224, 487-499 (1992)), Canonical
(Morea, et al., Antibody modeling: implications for engineering and
design, Methods 20, 267-279 (2000) and VH-VL interface (Chothia,
C., Novotny, J., Bruccoleri, R., & Karplus, M. Domain
association in immunoglobulin molecules. The packing of variable
domains. J. Mol. Biol. 186, 651-663 (1985)) residues and with CDRs
of identical canonical class and/or length. The identity of each
member of this library to individual aligned residues of the mouse
antibody was calculated using the program. Those human sequences
with FR sequence most identical to the mouse FR were identified,
producing an initial shortlist of human "acceptor" sequences. Those
sequences with most identity to the mouse antibody, at Vernier,
Canonical and VH-VL Interface (VCI) residues, were also calculated.
Differences at these positions between human and mouse were
classified into conservative and non-conservative substitutions, so
that the best framework choice would have the lowest number of
non-conservative VCI differences from LT1002. The CDR loops L3 and
H1 of LT1002 could be classified into canonical structures. These
L3 and H1 structures were used to select human antibody FRs with
identical canonical structures. For unclassified CDRs, an attempt
was made to select human frameworks with CDR lengths identical to
the mouse antibody. The rationale is that CDR loop structures are
dependent not only on the CDR loop sequence itself, but also on the
underlying framework residues (canonical residues). Therefore a
human framework with matching canonical CDR structures and/or CDR
lengths is likely to hold the grafted mouse CDRs in the most
appropriate orientation to maintain antigen binding affinity. This
was achieved for all CDRs except CDR H3, by the choice of human
framework sequences. Additionally, frameworks with unusual cysteine
or proline residues were excluded where possible. These
calculations were performed separately for the heavy and light
chain sequences. Finally, individual sequence differences,
throughout the framework region, in the best matching sequences
were compared. Of the human antibodies that best fit the above
comparative calculations, the antibodies AY050707 and AJ002773 were
selected as the most appropriate human framework provider for the
light chain and the heavy chain respectively.
The second step was to generate a molecular model of the variable
regions of LT1002 and to identify FR residues which might affect
antigen binding but were not included in the group of Vernier,
Canonical and Interface residues. Many structural features of the
graft donor and acceptor variable domains were examined in order to
better understand how various FR residues influence the
conformation of the CDR loops and vice versa. Non-conserved FR
residues in LT1002 that were likely to impact the CDRs were
identified from the Vernier and Canonical definitions (see above)
and thus several residues of the human FR were restored to the
original murine amino acids (backmutated).
b. Mutagenesis
Mutations within the variable domain sequences were created using
the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Catalog
#200524). Individual reactions were carried out with 50 ng of
double-stranded DNA template, 2.5 U of PfuUltre HF DNA polymerase
and its corresponding buffer (Stratagene, Catalog #200524), 10 mM
dNTP mix and 125 ng of each of the mutagenic oligonucleotides
resuspended in 5 mM Tris-HCl (pH 8.0), and 0.1 mM EDTA. The initial
denaturation was carried out at 95.degree. C. for 30 s, followed by
16 cycles of amplification: 95.degree. C. for 30 s, 55.degree. C.
for 60 s and 68.degree. C. for 8 min. Following temperature
cycling, the final reaction was then digested with DpnI digest at
37.degree. C. for 1 h to remove methylated parental DNA. The
resultant mutant was transformed into competent XL 1-Blue E. coli
and plated on LB-agar containing 50 .mu.g/ml Ampicillin. The
colonies were then checked by sequencing. Each of the mutants were
then cultured in 1 liter shake flasks and purified using the
EndoFree Plasmid Purification Kit from Qiagen, catalog #12362.
c. Generation of the Humanized Antibody Variants
A mouse-human chimeric antibody (chMAb S1P) was constructed by
cloning the variable domains of LT1002 into a vector that contained
the human constant regions of the kappa and heavy chains to allow
expression of the full length antibody into mammalian cells. The
generation of the humanized heavy chain was the result of the graft
of the Kabat CDRs 1, 2 and 3 from LT1002 V.sub.H into the acceptor
framework of AJ002773. The nearest germ line gene to AJ002773 was
VH5-51, whose leader sequence was incorporated, as a leader
sequence, into the humanized heavy chain variant. The protein
sequence of pATH200, the first humanized version of LT1002 V.sub.H,
with the VH5-51 leader sequence, is shown in Table 4. In the case
of the V.sub.H domain of LT1002, residues at position 2, 27, 37,
48, 67 and 69 were Vernier residues or at the interface of the
V.sub.H and V.sub.L domains and likely to influence CDR
orientation. Position 37 appeared to be critical for the interface
between the V.sub.H and V.sub.L domains. The residues at these
positions in the human framework were backmutated with the murine
residue found at the corresponding position. The mutations, V37M,
M481 and Y27F, were tested individually. One version (pATH205)
contained all 3 mutations together with V67A plus 169 L and another
version (pATH206) contained all 5 mutations plus V2A.
The generation of the humanized light chain was the result of the
graft of the Kabat CDRs 1, 2 and 3 from LT1002 V.sub.L into the
acceptor framework of AY050707. The nearest germ line gene to
AY050707 was L11, whose leader sequence was incorporated into the
humanized light chain construct. The protein and DNA sequences of
pATH300 (LT1002 light chain) are SEQ ID NO: 17 and 28, respectively
(see Table 4 for amino acid sequence). In the case of V.sub.L, four
non-conserved Vernier positions 4, 36, 49, 64 were selected for
backmutation to murine residues as they are involved in supporting
the structure of the CDR loops. Inspection of the molecular model
of LT1002 suggested that Tyr 67 is close to the CDR surface and
oriented towards the antigen binding plane and could interact with
S1P. Therefore the S67Y backmutation was also added to later
humanized versions. Two mutations were introduced separately to
generate two versions containing either Y49S or Y36F. Several
versions were created with the following combinations of mutations:
(Y49S, F4V), (Y49S, Y36F), (Y49S, Y36F, F4V), (Y49S, G64S), (Y49S,
Y36F, F4V, G64S), (Y49S, Y36F, F4V, G64S, S67Y), (Y49S, G64S,
S67Y).
d. Selection of the Humanized Lead Candidates
The variable regions of the basic grafted versions (pATH 200 and
pATH 300) and all the variants containing backmutations were cloned
into expression vectors containing the human V.sub.H or V.sub.L
constant regions. All the humanized variants were produced in
mammalian cells under the same conditions as the chimeric (chMAb)
antibody and were tested for binding to S1P by ELISA. The yield was
approximately 10-20 mg/l for the humanized variants and 0.3-0.5
mg/l for chMAb S1P. SDS-PAGE under reducing conditions revealed two
bands at 25 kDa and 50 kDa with high purity (>98%), consistent
with the expected masses of the light and heavy chains. A single
band was observed under non-reducing conditions with the expected
mass of .about.150 k. chMAb was used as a standard in the humanized
antibody binding assays because it contained the same variable
regions as the parent mouse antibody and bore the same constant
regions as the humanized antibodies and therefore could be detected
using the same ELISA protocol.
The initial humanized antibody, in which the six murine CDRs were
grafted into unmutated human frameworks, did not show any
detectable binding to S1P (FIG. 11). The kappa light chain
containing the 4 backmutations (Y49S, Y36F, F4V and G64S), in
association with chimeric heavy chain, exhibited suboptimal binding
to S1P as measured by ELISA. The incorporation of an additional
mutation at position Y67 significantly improved the binding.
Version pATH308 which contained backmutations Y49S, Y36F, F4V, G64S
and S67Y and version pATH309 which contained the backmutations
Y49S, G64S and S67Y, in association with chimeric heavy chain, both
generated antibodies which bound S1P similarly to the chimeric
antibody as determined by ELISA. The 2 mutations Y36F and F4V were
not considered necessary backmutations from the viewpoint of S1P
binding. The engineering of 3 to 5 backmutations in the V.sub.L
framework was required to restore activity.
The incorporation of the Vernier backmutation V37M into the human
framework of the heavy chain, in association with the chimeric
light chain, was sufficient to restore a binding behavior similar
to the chimeric antibody (FIG. 11).
In summary, humanization of the LT1002 V.sub.H domain required only
one amino acid from the murine framework sequence whereas the
murine V.sub.L framework domain, three or five murine residues had
to be retained to achieve binding equivalent to the murine parent
LT1002.
e. Optimization of a Humanized Lead Candidate
The murine anti-S1P antibody contains a free cysteine residue in
CDR2 (Cys50) of the heavy chain that could potentially cause some
instability of the antibody molecule. Using site directed
mutagenesis, variants of pATH201 were created with substitution of
the cysteine residue with alanine (huMAbHCcysalaLC.sub.3)
(pATH207), glycine (huMAbHCcysalaLC.sub.3), serine
(huMAbHCcysserLC.sub.3), and phenylalanine (huMAbHCcyspheLC.sub.3).
The cysteine mutant heavy chain was also tested with the humanized
light chain (pATH 308) containing 5 backmutations
(huMAbHCcysalaLC.sub.5=LT1009). The variants were expressed in
mammalian cells and then characterized in a panel of in vitro
assays. Importantly, the expression rate of the humanized variants
was significantly higher than for chMAb S1P.
f. In-Depth Characterization of the Humanized Lead Candidate
i. Specificity. The humanized variants were tested for specificity
in a competitive ELISA assay (FIG. 1) against S1P and several other
biolipids. This assay has the added benefit to allow for epitope
mapping. The humanized antibody LT1009 demonstrated no
cross-reactivity to sphingosine (SPH), the immediate metabolic
precursor of S1P, or LPA (lysophosphatidic acid), an important
extracellular signaling molecule that is structurally and
functionally similar to S1P. Moreover, rhuMAb S1P did not recognize
other structurally similar lipids and metabolites, including
ceramide (CER), ceramide-1-phosphate (C1P). However as expected
LT1009 did cross react with sphingosyl phosphocholine (SPC), a
lipid in which the free phosphate group of S1P is tied up with a
choline residue. Importantly, all the humanized variants exhibited
a specificity profile comparable to the mouse antibody.
ii. Binding affinity. Biacore measurements of IgG binding to a S1P
coated chip showed that the variants LT1004 or LT1006 exhibited
binding affinity in the low nanomolar range similar to chMAb S1P as
shown in FIG. 11. The humanized variants LT1007 and LT1009 in which
the cysteine residue was replaced with alanine exhibited a binding
affinity in the picomolar range similar to the murine parent LT1002
(Sphingomab.TM.).
iii. Stability. The humanized variants were tested for stability
after challenge at high temperature. The approximate midpoints of
the thermal unfolding transitions (T.sub.M) were determined for
every humanized variant by subjecting the supernatants to
temperatures ranging from 60 to 74.degree. C. These temperatures
were chosen based on the denaturation profile observed for the
murine antibody molecule after thermochallenging between a broad
range of temperatures between 50 and 80.degree. C. The binding
properties of each variant were determined before and after
thermochallenge. The murine antibody exhibited a T.sub.M of
65.degree. C. The variant huMAbHCcysalaLC.sub.5 (LT1009) exhibited
superior T.sub.M compared to all other variants. Table 7 shows the
lead humanized candidates and their characteristics.
TABLE-US-00007 TABLE 7 Lead humanized S1P mAb candidates and
characteristics The number of mutations in the heavy and light
chains are indicated. The description column gives the identity of
the heavy and light chains. Mutations in Mutations in the Heavy the
Light In vitro Activity Chain Chain Binding Frame- Frame- Affinity
Specificity mAb Description CDR work CDR work (K.sub.D1) (ELISA)
LT1002 Murine mAb N/A N/A N/A N/A 0.026 .+-. 0.000 nM High
Sphingomab LT1004 HuHCLC.sub.3 0 1 0 3 1.060 .+-. 0.010 nM High
pATH201HC pATH309LC LT1006 HuHCLC.sub.5 0 1 0 5 0.690 .+-. 0.010 nM
High pATH201HC pATH308LC LT1007 HuHCcysalaLC.sub.3 1 1 0 3 0.0414
.+-. 0.0004 nM pATH207HC pATH309LC LT1009 HuHCcysalaLC.sub.5 1 1 0
5 0.056 .+-. 0.001 nM High pATH207HC pATH308LC
iv. Sequences
As with naturally occurring antibodies, LT1009 includes three
complementarity determining regions (each a "CDR") in each of the
two light chain polypeptides and each of the two heavy chain
polypeptides that comprise each antibody molecule. The amino acid
sequences for each of these six CDRs is provided immediately below
("VL" designates the variable region of the immunoglobulin light
chain, whereas "VH" designates the variable region of the
immunoglobulin heavy chain):
TABLE-US-00008 CDR1 VL: ITTTDIDDDMN [SEQ ID NO: 10] CDR2 VL:
EGNILRP [SEQ ID NO: 11] CDR3 VL: LQSDNLPFT [SEQ ID NO: 12] CDR1 VH:
DHTIH [SEQ ID NO: 13] CDR3 VH: GGFYGSTIWFDF [SEQ ID NO: 15] CDR2
VH: AISPRHDITKYNEMFRG [SEQ ID NO: 31]
The nucleotide and amino acid sequences for the heavy and light
chain polypeptides of LT1009 are listed immediately below:
TABLE-US-00009 LT1009 HC amino acid sequence of the variable domain
[SEQ ID NO: 32]: 1 mewswvflff lsvttgvhse vqlvqsgaev kkpgeslkis
cqsfgyifid 51 htihwmrqmp gqglewmgai sprhditkyn emfrgqvtis
adkssstayl 101 qwsslkasdt amyfcarggf ygstiwfdfw gqgtmvtvss LT1009
LC amino acid sequence of the variable domain [SEQ ID NO: 33]: 1
msvptqvlgl lllwltdarc ettvtqspsf lsasvgdrvt itcitttdid 51
ddmnwfqqep gkapkllise gnhlrpgvps rfsssgygtd ftltisklqp 101
edfatyyclq sdnlpftfgq gtkleik LT1009 HC nucleotide sequence [SEQ ID
NO: 34]: 1 aagcttgccg ccaccatgga atggagctgg gtgttcctgt tctttctgtc
51 cgtgaccaca ggcgtgcatt ctgaggtgca gctggtgcag tctggagcag 101
aggtgaaaaa gcccggggag tctctgaaga tctcctgtca gagttttgga 151
tacatcttta tcgaccatac tattcactgg atgcgccaga tgcccgggca 201
aggcctggag tggatggggg ctatttctcc cagacatgat attactaaat 251
acaatgagat gttcaggggc caggtcacca tctcagccga caagtccagc 301
agcaccgcct acttgcagtg gagcagcctg aaggcctcgg acaccgccat 351
gtatttctgt gcgagagggg ggttctacgg tagtactatc tggtttgact 401
tttggggcca agggacaatg gtcaccgtct cttcagcctc caccaagggc 451
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac 501
agcggccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg 551
tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 601
gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 651
ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc 701
ccagcaacac caaggtggac aagagagttg gtgagaggcc agcacaggga 751
gggagggtgt ctgctggaag ccaggctcag cgctcctgcc tggacgcatc 801
ccggctatgc agtcccagtc cagggcagca aggcaggccc cgtctgcctc 851
ttcacccgga ggcctctgcc cgccccactc atgctcaggg agagggtctt 901
ctggcttttt ccccaggctc tgggcaggca caggctaggt gcccctaacc 951
caggccctgc acacaaaggg gcaggtgctg ggctcagacc tgccaagagc 1001
catatccggg aggaccctgc ccctgaccta agcccacccc aaaggccaaa 1051
ctctccactc cctcagctcg gacaccttct ctcctcccag attccagtaa 1101
ctcccaatct tctctctgca gagcccaaat cttgtgacaa aactcacaca 1151
tgcccaccgt gcccaggtaa gccagcccag gcctcgccct ccagctcaag 1201
gcgggacagg tgccctagag tagcctgcat ccagggacag gccccagccg 1251
ggtgctgaca cgtccacctc catctcttcc tcagcacctg aactcctggg 1301
gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 1351
tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1401
gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 1451
tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg 1501
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1551
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat 1601
ctccaaagcc aaaggtggga cccgtggggt gcgagggcca catggacaga 1651
ggccggctcg gcccaccctc tgccctgaga gtgaccgctg taccaacctc 1701
tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat 1751
cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1801
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc 1851
ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct 1901
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1951
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac 2001
gcagaagagc ctctccctgt ctccgggtaa atag LT1009 HC amino acid sequence
[SEQ ID NO: 35]: 1 mewswvflff lsvttgvhse vqlvqsgaev kkpgeslkis
cqsfgyifid 51 htihwmrqmp gqglewmgai sprhditkyn emfrgqvtis
adkssstayl 101 qwsslkasdt amyfcarggf ygstiwfdfw gqgtmvtvss
astkgpsvfp 151 lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv
htfpavlqss 201 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvap
ellggpsvfl 251 fppkpkdtlm isrtpevtcv vvdvshedpe vkfnwyvdgv
evhnaktkpr 301 eeqynstyrv vsvltvlhqd wlngkeykck vsnkalpapi
ektiskakgq 351 prepqvytlp psreemtknq vsltclvkgf ypsdiavewe
sngqpennyk 401 ttppvldsdg sfflyskltv dksrwqqgnv fscsvmheal
hnhytqksls 451 lspgk LT1009 LC nucleotide sequence [SEQ ID NO: 36]:
1 aagcttgccg ccaccatgtc tgtgcctacc caggtgctgg gactgctgct 51
gctgtggctg acagacgccc gctgtgaaac gacagtgacg cagtctccat 101
ccttcctgtc tgcatctgta ggagacagag tcaccatcac ttgcataacc 151
accactgata ttgatgatga tatgaactgg ttccagcagg aaccagggaa 201
agcccctaag ctcctgatct ccgaaggcaa tattcttcgt cctggggtcc 251
catcaagatt cagcagcagt ggatatggca cagatttcac tctcaccatc 301
agcaaattgc agcctgaaga ttttgcaact tattactgtt tgcagagtga 351
taacttacca ttcactttcg gccaagggac caagctggag atcaaacgta 401
cggtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg 451
aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag 501
agaggccaaa gtacagtgga aggtggataa cgccctccaa tcgggtaact 551
cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 601
agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta 651
cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct 701
tcaacagggg agagtgttag LT1009 LC amino acid sequence [SEQ ID NO:
37]: 1 msvptqvlgl lllwltdarc ettvtqspsf lsasvgdrvt itcitttdid 51
ddmnwfqqep gkapkllise gnhlrpgvps rfsssgygtd ftltisklqp 101
edfatyyclq sdnlpftfgq gtkleikrtv aapsvfifpp sdeqlksgta 151
svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 201
lskadyekhk vyacevthqg lsspvtksfn rgec
Example 13
Humanized S1P mAb Production and Purification
This example describes the production of a recombinant humanized
monoclonal antibody (LT1009; Sonepcizumab.TM.) that binds with high
affinity to the bioactive lipid sphingosine-1-phosphate (S1P).
LT1009 is a full-length IgG1k isotype antibody composed of two
identical light chains and two identical heavy chains with a total
molecular weight of 150 kDa. The heavy chain contains an N-linked
glycosylation site. The nature of the oligosaccharide structure has
not yet been determined but is anticipated to be a complex
biantennary structure with a core fucose. The nature of the
glycoform that will be predominant is not known at this stage. Some
C-terminal heterogeneity is expected because of the presence of
lysine residues in the constant domain of the heavy chain. The two
heavy chains are covalently coupled to each other through two
inter-chain disulfide bonds, which is consistent with the structure
of a human IgG1.
LT1009 was originally derived from a murine monoclonal antibody
(LT1002; Sphingomab.TM.) that was produced using hybridomas
generated from mice immunized with S1P. The humanization of the
murine antibody involved the insertion of the six murine CDRs in
place of those of a human antibody framework selected for its
structure similarity to the murine parent antibody. A series of
substitutions were made in the framework to engineer the humanized
antibody. These substitutions are called back mutations and replace
human with murine residues that are play a significant role in the
interaction of the antibody with the antigen. The final humanized
version contains one murine back mutation in the human framework of
variable domain of the heavy chain and five murine back mutations
in the human framework of the variable domain of the light chain.
In addition, one residue present in the CDR #2 of the heavy chain
was substituted to an alanine residue. This substitution was shown
to increase stability and potency of the antibody molecule.
The humanized variable domains were cloned into the Lonza's GS gene
expression system to generate the plasmid pATH1009. This expression
system consists of an expression vector carrying the constant
domains of the antibody genes and the selectable marker glutamine
synthetase (GS). GS is the enzyme responsible for the biosynthesis
of glutamine from glutamate and ammonia. The vector carrying both
the antibody genes and the selectable marker is transfected into
proprietary Chinese hamster ovary (CHOK1SV) host cell line adapted
for growth in serum-free medium and provides sufficient glutamine
for the cell to survive without exogenous glutamine. In addition,
the specific GS inhibitor, methionine sulphoximine (MSX), is
supplemented in the medium to inhibit endogenous GS activity such
that only the cell lines with GS activity provided by the vector
can survive. The transfected cells were selected for their ability
to grow in glutamine-free medium in the presence of MSX and
isolates were selected for high level of secretion of active
LT1009. Material for toxicology studies and clinical development
were then produced for tox and clinical development.
ATCC deposits: E. coli StB12 containing the pATH1009 plasmid has
been deposited with the American Type Culture Collection (ATCC,
P.O. Box 1549, Manassas, Va. 20108, USA) (deposit number PTA-8421;
received by the ATCC on 10 May 2007). CHO cell line LH1 275, which
contains the pATH 1009 vector has also been deposited with the
American Type Culture Collection (deposit number PTA-8422; received
on 10 may 2007).
Example 14
Manufacturing of Humanized mAb
Typically, the production process involves three stages: seed
train, inoculum train, and the production culture. All stages use
serum-free reagents and low protein cell culture growth medium. To
initiate a seed train, cells from the working cell bank are used;
cells are subcultured every three to four days and after a
prescribed period in the seed train culture, the inoculation train
is initiated. The non-selective medium (MTX-freee medium) is
preferably used to expand the cell for introduction into the
production stage. The cells are expanded by serial sub-cultivation
into vessels of increasing volume. At a certain number of days in
the inoculum train, the production stage is initiated. The
production culture is performed in a bioreactor of volume of 200 L,
400 L, 2000 L, or 20000 L. An example of a bioreactor is described
below.
Example of bioreactor: Scale-up from the 2 L bioreactors will
proceed first to a Applikon 15 L stirred tank, then sequentially to
a 50 L bioreactor, a 200 L bioreactor and finally a 2000 L
bioreactor (all built to same scale). The characteristics of these
tanks are as follows:
Manufacturer: ABEC, Inc.
Fabricated to ASME, Section VIII Pressure Vessel Code; Contact
surfaces are 316L SS.
Bottom offset drive, ABEC design
Lowshear impellor 316LSS, polished to 15-20 microinch and
passivated. Diameter .about.1/2 of vessel diameter.
Controls: Allen Bradley Control Logic PLC, with Versa view operator
interface.
Agitation: Allen Bradley sensor, A-B PLC control of output for VFI
for RPM control
Temperature: Dual control 100 ohm platinum RTD sensor, A-B PLC
control heat, cold and steam valves w/recirculation pump. Automatic
sterilization cycle with bioreactor empty.
pH: Ingold sensor, gel filled, pressurizable, A-B PLC control of
CO2 to sparge.
Dissolved oxygen: Ingold polarographic electrode sensor, A-B PLC
control of O2 sparge.
Air and gas flow: Sensors are Four Brooks Thermal Mass Flowmeters
for air, O2, N2 and CO2 sparging, a Brooks thermal mass is also
supplied for air overlay, A-B PLC control of gas flows for pH auto,
DO auto or manual control of all gas flow through A-B PLC.
Vessel Pressure: Sensor is a Rosemount sanitary diaphragm type
transducer, control is A-B PLC control of transducer with back
pressure control valve.
Programmable Logic Controller (PLC): Allen Bradley Control Logix
System for sequential loop control of processes as indicated.
Software: PLC programming utilizes Rockwell Software (Allen
Bradley) RS Logix 5000.
Human Machine Interface (HMI): Local operator interface is on an
Allen Bradley HMI Verso View Industrial computer with integrated
FPD/touch screen entry communicating to the PLC via Ethernet.
Software is Rockwell Software RS View 32.
Example of Production Process.
Stirred stainless bioreactor with control of temperature, dissolved
oxygen and pH.
Seeding density is determined for optimal yield.
Serum-free medium is typically utilized.
Typically a fed-batch process.
Typically with a temperature shift.
Duration of culture in the bioreactor expected to be 8 to 14
days.
Viability at time of harvest to be defined.
Harvest will be clarified by filtration.
Harvest will be stored at 2-8.degree. C. following clarification
and prior to purification.
Example 15
Large-Scale Purification of Humanized mAb
The drug substance purification process typically consists of four
steps: protein A chromatography, anion exchange chromatography (Q
sepharose), cation exchange chromatography (CM sepharose), and
ultrafiltration/diafiltration (UF/DF). The affinity column is the
generally the first step after harvest and clarification. This
column typically utilizes an immobilized protein A resin. This
affinity step purifies the antibody with respect to host cell
proteins and DNA. In order to inactivate potential viruses, the
eluate is typically subjected to a virus inactivation process
followed by an anion exchange chromatographic step to reduce host
cell proteins, DNA, protein A, and potential viruses. Next, a
cation exchange chromatographic step is typically used to further
reduce the residual amounts of host cell proteins and antibody
aggregates. Finally, the pool is then diafiltered and further
concentrated.
Representative Purification Process:
Harvest may be concentrated and buffer-exchanged prior to Protein A
column. The next step in the process is Protein A column affinity
chromatography. The bound antibody is eluted with a low pH buffer.
The Protein A eluate is held for a time in order to inactivate
viruses.
The next step in the process can be an ion-exchange chromatography
on a Q(+) column under conditions in which the antibody product
flows through and contaminants, such as DNA and host cell proteins
bind to the column resin.
The next step in the process can be a second ion-exchange
chromatography on an S(-) column under conditions in which
contaminants flow through the column. A hydrophobic interaction
column step may be used in place of the S(-) column step. The next
step in the process is likely to be a nanofiltration virus removal
step, using a DV20 or Planova filter. The product flows through the
filter. The final steps in the process are diafiltration into the
final drug substance formulation buffer and ultrafiltration to
achieve the target protein concentration.
Example 16
Biological Activity of Humanized Variants of a Murine Anti-S1P
Antibody
In Vitro Cell Assays
The humanized antibodies were tested for their ability to alter
tumor cell survival in presence of chemotherapeutic agents as shown
in FIG. 12. SKOV3 tumor cells were exposed to Taxol, a
chemotherapeutic agent that induces tumor cell death by activation
of the apoptotic executioner, caspase-3. S1P was able to decrease
Taxol-induced caspase-3 activation and/or cell death compared to
the control non-treated cells. Apoptosis assays were performed as
recommended by the manufacturer (Promega, Cat. No G7792). Briefly,
A549 cells (2500 cells per well) were seeded into 96-well plates
and allowed to grow to 80% confluence prior to treatment. The cells
were then treated with and without 0.1-1 .mu.M Paclitaxel (Sigma,
Cat. No T 7409), 0.1-1 .mu.M S1P and 1 .mu.g/mL of the anti-S1P
mAb, in McCoy's media for 48 hrs. After 48 hrs, the caspase assay
buffer was added to the cells. Caspase-3 activity in the
supernatant was measured by Apo-One Homogeneous Caspase-3/7 Assay
kit (Promega, Cat. No G7792) according to the manufacturer's
protocol. Caspase-3/7 activity is expressed as the fold increase in
fluorescence signal with respect to vehicle treated cells.
Caspase-3 activation was increased by the addition of anti-S1P mAb
in presence of S1P, suggesting that the protective anti-apoptotic
effect of S1P was eliminated by selective absorption of S1P by the
antibody. Both humanized antibody variants, huMAbHCLC.sub.3
(LT1004) and huMAbHCLC.sub.5 (LT1006), exhibited superior activity
compared to LT1002. In parallel, all the variants were tested for
their effects on S1P-induced cytokine release from cancer cells.
S1P is known to elicit significant release of IL-8 into the
cell-conditioned media from cancer cells. Addition of the mouse
control anti-S1P mAb reduced IL-8 release from ovarian cancer cells
in a concentration-dependent manner. The two humanized variants
huMAbHCcysalaLC.sub.3 (LT1007) and huMAbHCcysalaLC.sub.5 (LT1009)
exhibited greater reduction of IL-8 release compared to
HuMAbHCLC.sub.3 (LT1004) and huMAbHCLC.sub.5 (LT1006).
Example 17
In Vivo Efficacy of Murine mAb (Sphingomab) Vs, Humanized mAb
(Sonepcizumab) in an Animal Model of Neovascularization
Choroidal neovascularization (CNV) refers to the growth of new
blood vessels that originate from the choroid through a break in
the Bruch membrane into the sub-retinal pigment epithelium
(sub-RPE) or subretinal space in the eye. CNV is a major cause of
visual loss in macular degeneration and other ocular conditions. A
mouse model of CNV is used in this example for evaluation of mAbs
against S1P.
The humanized antibody variants and the murine antibody were
compared for their ability to inhibit neo-vascularization in the
CNV animal model of AMD as shown in FIG. 13. Mice were administered
0.5 ug twice (Day 0 and Day 6) of the murine (Mu; LT1002), the
humanized variants [LC3 (LT1004), LC5 (LT1006), HCcysLC3 (LT1007)
and HCcysLC5 (LT1009)] or the nonspecific mAb (NS) by intravitreal
administration and then subjected to laser rupture of Bruchs
membrane. Mice were sacrificed 14 days post laser surgery. Control
mice were treated with aqueous buffer (PBS) or an isotype-matched
non-specific antibody. Three of the humanized variants inhibited
angiogenesis essentially equivalently to the murine antibody as
assessed by measurement of CNV area. CNV lesion volumes are
represented as means.+-.SEM. The humanized variant containing 5
backmutations in the light chain and with a cysteine mutation in
CDR2 of the heavy chain (huMAbHCcysLC.sub.5; LT1009) markedly
suppressed neovascularization. This difference was highly
statistically significant.
For the induction of CNV, mice were anesthetized with a mixture of
ketamine (14 mg/kg) and xylazine (30 mg/kg) in sterile saline
administered intraperitoneally at a dose of 5 .mu.L per 20 g of
body weight. Their pupils were then dilated with one drop each of
ophthalmic tropicamide (0.5%) and phenylephrine (2.5%). An argon
green ophthalmic laser (Oculight GL 532 nm, Iridex Corporation,
Mountain View, Calif.) coupled to a slit lamp set to deliver a 100
msec pulse at 150 mW with a 50 .mu.m spot size will then be used to
rupture Bruch's membrane in three quadrants of the right eye
located approximately 50 .mu.m from the optic disc at relative 9,
12 and 3'oclock positions. The left eye served as uninjured control
in all cases.
The morphometric and volumetric CNV lesions were measured as
follows. Two weeks after laser induction of CNV, the animals were
euthanized by overdose of ketamine-xylazine mixture, then undergo
whole body perfusion via cardiac puncture with 6 ml 4%
paraformaldehyde in PBS, pH 7.5, (fixative) as previously described
(Sengupta et al., 2003). The eyes will then be enucleated,
punctured with a 27 g needle 1 mm posterior to the limbus, and
immersed in fixative for 1 hr at room temperature, then washed
2.times. by immersion in PBS for 30 min. The eyes will then be
dissected to isolate the posterior segment consisting of the
retinal pigment epithelium, the choriocapillaris and the sclera.
This tissue was then permeabilized and reacted with
rhodamine-conjugated R. communis agglutinin I (Vector Laboratories,
Burlingame, Calif.) to detect the CNV lesion as previously
described (Sengupta et al., 2003; Sengupta et al., 2005). The
posterior cups was then cut with 4-7 radial slices, and mounted
flat on microscope slides with a drop of Vectashield anti-fade
medium (Vector Laboratories, Burlingame, Vt.) for digital image
capture by epifluorescence Zeiss Axioplan 2 with RGB Spot
high-resolution digital camera) and laser scanning confocal
microscopy (BioRad MRC 1024, BioRad Corporation, Temecula,
Calif.).
Captured digital images were evaluated morphometrically using
ImageJ software (Research Services Branch, National Institutes of
Health, Bethesda, Md.). Images were split into separate RGB
channels for analysis of the red and green channels as follows: 1)
a calibration for the specific objective and microscope was applied
to set the pixel-to-length ratio; 2) a threshold was applied using
the Otsu algorithm; 3) images will be made binary; 4) a
region-of-interest (ROI) was outlined to include the entire lesion
area; 5) a particle analysis was performed to quantify the pixel
area above the threshold level within the ROI. For volumetric
analysis, the process was similar to that described above, except
that a z-series capture was used. The sum of lesion area throughout
the z-series was then multiplied by the z thickness (typically 4
.mu.m) to obtain the lesion volume.
Drug products tested in this model were LT1002 (murine mAb to S1P;
Sphingomab.TM.); LT1004 (humanized mAb), LT1006 (humanized mAb),
LT1007 (humanized mAb) and LT1009 (humanized mAb;
Sonepcizumab.TM.). Also included were saline vehicle and
non-specific antibody (NSA) controls. As shown in FIG. 13, both the
murine mAb LT1002 (Sphingomab.TM.) and the humanized mAb LT1009
(Sonepcizumab.TM.) significantly decreased lesion size in this
mouse model of CNV. All mAbs tested showed approximately 80-98%
reduction of lesion size, which was significant (p<0.001 vs.
saline) in all cases. In addition, LT1007 and LT1009 also showed
significant inhibition (p<0.05) compared to non-specific
antibody control. Percent inhibition of lesion size was
approximately 80% for LT1002 (murine), 82% for LT1004 (humanized),
81% for LT1006 and 99% for LT1009. Thus, LT1009 was the humanized
mAb variant most active in this in vivo model of
neovascularization.
Example 18
Determination of the Sonepcizumab Dose Response
Mice (n=10) received a single, bilateral intravitreal injection of
escalating doses of sonepcizumab (0.05, 0.5, 1.0 or 3.0 .mu.g/eye)
or a high dose nonspecific (NS) antibody (3.0 .mu.g/eye) one day
prior to laser-induced rupture of Bruch's membrane. Fourteen days
after laser rupture, mice were anesthetized and perfused with
fluorescein-labeled dextran and choroidal flatmounts were prepared
for analysis of CNV lesion size.
In this study, the effect of sonepcizumab dose amount and dose
interval on CNV inhibition were examined using another validated
method of quantifying CNV area in which animals were perfused with
fluorescein-labeled dextran just before sacrifice. Sonepcizumab
induced a dose-dependent reduction in the area of CNV resulting in
a maximal inhibition of approximately 50%, at a dose of 3.0
.mu.g/eye. This reduction was significant (p<0.0001 compared to
non-specific antibody control using an unpaired t-test). In the
dosing frequency study, similar efficacy was observed between
groups treated with Sonepcizumab at a single timepoint (day 0) or
at multiple timepoints (days 0 and 7) over the 14-day study.
The maximal inhibition of approximately 50% seen with Sonepcizumab
treatment (3.0 ug/eye) compares favorably with previously published
data in the same model and conducted by the same investigator
demonstrating the reduction in CNV area by the VEGF-Trap (4.92
.mu.g/eye). Saishin, et al. J Cell Physiol, 2003. 195(2): p. 241-8.
"Traps" (Regeneron Pharmaceuticals, Inc.) are fusions between two
distinct receptor components and the Fc region of an antibody
molecule called the Fc region and the VEGF-Trap is being pursued
for ocular disease and cancer by Regeneron. A comparison of these
two independent studies reveals that the reduction in CNV lesion
size by Sonepcizumab was 20 percentage points greater than that
observed with the VEGF-Trap. Thus, these data not only confirm our
preliminary findings regarding the ability of an anti-S1P therapy
to reduce lesion formation in murine model of CNV, but they also
demonstrate the increased efficacy of the humanized antibody,
sonepcizumab, to inhibit CNV lesion formation and provide insight
into an anti-permeability effect.
Example 19
Efficacy of Sonepcizumab in Reducing the Development of Retinal
Neovascularization in a Murine Model of Retinopathy of
Prematurity
C57BL/6 mice (n=7) were placed in 75% oxygen at day 7 of life and
at day 12 of life were returned to room air and given an
intraocular injection of 31 g of sonepcizumab in one eye and
vehicle in the contralateral eye. At day 17, the mice received an
intraocular injection of anti-PECAM antibody labeled with FITC and
after 8 hours, mice were euthanized and eyes were removed and fixed
in PBS-buffered formalin at room temperature for 5 hours. Retinas
were dissected and washed with phosphate-buffered saline containing
0.25% Triton X-100 and whole mounted. Slides were viewed with a
Nikon Fluorescence Microscope and the area of retinal NV per retina
was measured by image analysis.
Consistent with the reduction in CNV observed in the murine laser
rupture model, we also observed a dramatic reduction in CNV in a
murine model of retinopathy of prematurity (ROP). Intravitreal
administration of Sonepcizumab (3.0 .mu.g/eye) resulted in a nearly
4-fold reduction in retinal neovascularization compared to saline
control. These data confirm the efficacy of sonepcizumab to inhibit
pathological ocular angiogenesis in both the retinal and choroidal
vascular beds whether induced via ischemia or rupture of Bruch's
membrane.
Example 20
Effect of Sonepcizumab on VEGF-Induced Angiogenesis in a Matrigel
Plus Assay
Neovascularization in vivo was performed using the GFR Matrigel
plug assay as described in Staton, et al., Int J Exp Pathol, 2004.
85(5): p. 233-48. 4-6 week old nu/nu mice were injected in the left
flank with 500 uL of ice-cold GFR Matrigel. The GFR Matrigel was
injected either alone (control) or after addition of 10 ug/mL VEGF
supplemented with 100 ug/ml heparin. Groups consisted of 3 animals
for control and sonepcizumab treatment. Animals were treated with
the saline or sonepcizumab (10 mg/kg) I day prior to the
implantation of GFR Matrigel and doses were administrated i.p.
every 72 hrs for the duration of the experiment. After 12 days
animals were sacrificed; the plugs were excised and immediately
fixed in zinc and formalin-free fixative overnight, embedded in
paraffin and sectioned (5 um). Paraffin-embedded sections were then
stained for CD31 (Pharmingen). Images (9 images per section, 3
sections per plug) were taken by digital camera at 20.times.
magnification and the CD31 positive staining was then quantified by
PhotoShop 6.0 program and expressed as angiogenesis score
(pixel.sup.2) by ImageJ.
The anti-angiogenic effects of sonepcizumab were evident in this
Matrigel plug assay. As expected extensive neovascularization
(approx. 5.75.times. that seen in untreated control lacking VEGF or
sonepcizumab) was induced in the Matrigel plugs supplemented with
10 ug/ml VEGF. Importantly, systemic i.p. treatment with
sonepcizumab prior to Matrigel injection prevented nearly 80% of
this VEGF-stimulated increase in cellularity and microvessel
density. This reduction is significant (p<0.05 compared to VEGF
alone) and confirms the potent anti-angiogenic activity of
sonepcizumab when administered systemically to animals and strongly
suggest that sonepcizumab is capable of significantly inhibiting
VEGF induced angiogenesis. This finding is consistent with data
from Lpath's oncology program whereby that S1P antibody reduced
serum levels of several angiogenic factors, including VEGF, in a
murine orthotopic breast cancer model.
A primary component of blood vessel growth associated with AMD is
the recruitment of pericytes which ensheath and support the growing
endothelial tube. Jo, et al., Am J Pathol, 2006. 168(6): p.
2036-53. Transgenic mouse studies have shown that VEGF and PDGF-B
are the primary factors that stimulate infiltration and
differentiation of pericytes leading to blood vessel maturation and
stabilization. Guo, et al., Am J Pathol, 2003. 162(4): p. 1083-93;
Benjamin, L. E., I. Hemo, and E. Keshet, Development, 1998. 125(9):
p. 1591-8. Importantly, S1P promotes trans-activation of VEGF and
PDGF. Therefore, the ability of sonepcizumab to indirectly
neutralize these growth factors suggests that sonepcizumab could
prevent abnormal blood vessel growth during AMD.
Example 21
Sonepcizumab Significantly Reduces Vascular Leakage Following Laser
Rupture of Bruch's Membrane
The efficacy of sonepcizumab, administered to inhibit vascular
leakage (in addition to inhibiting neovascularization as shown
above) was evaluated in a murine model of laser rupture of Bruch's
membrane.
C57BL/6 mice (n=10) underwent laser rupture of Bruch's membrane in
3 locations in each eye and were given an intraocular injection of
3 .mu.g of sonepcizumab in one eye and vehicle in the contralateral
eye. At one week after laser rupture, the mice were given an
intraperitoneal injection of 12 .mu.l/g body weight of 1%
fluorescein sodium and were euthanized 5 minutes later. The eyes
were removed and fixed in PBS-buffered formalin at room temperature
for 5 hours. Then the retinas were dissected, washed, and incubated
with primary anti-PECAM-1. The retinas were then washed, incubated
with secondary antibody (goat anti-rat IgG conjugated with
rhodamine), and then flat mounted.
Quantification of CNV lesion area is measured by PECAM-1 staining.
Quantification of vascular leakage is measured by fluorescein
sodium staining. The total area of leakage from CNV=CNV+leakage
(green)-area of CNV (red). Values represent the mean.+-.SEM for
n=10 mice/group. The area of choroidal neovascularization (stained
by PECAM-1) was approximately 0.015 mm.sup.2 for animals treated
with LT1009 and approximately 0.03 mm.sup.2 for saline-treated
control animals. This is a 50% reduction in neovascularization
(p-0.018). The area of leakage from choroidal neovascularization
(stained by fluorescein) was approximately 0.125 mm.sup.2 for
animals treated with LT1009 and approximately 0.2 mm.sup.2 for
saline-treated control animals. This is approximately a 38%
reduction (p-0.017) in blood vessel leakage.
Representative immunohistochemical images of the reduction in
choroidal neovascularization and vascular leakage in mice treated
with 3.0 .mu.g/eye of Sonepcizumab or PBS control are consistent
with these results. Thus, in addition to reducing CNV, sonepcizumab
significantly reduced vascular leakage following laser rupture of
Bruch's membrane retinal edema, which plays a major role in the
loss of visual acuity, is associated with: (i) choroidal
neovasculature leakage in AMD and (ii) the breakdown of the
blood-retinal barrier in diabetes. Gerhardt, H. and C. Betsholtz,
Cell Tissue Res, 2003. 314(1): p. 15-23 Sonepcizumab reduces
pathological blood vessel formation in the eye as well as vascular
leakage that results in retinal edema. These findings are
consistent with the data generated from the CNV-area-quantification
experiment in which mice were perfused with fluorescein-labeled
dextran. CNV quantification via this method surely is affected by
vascular permeability. The highly favorable results argue for an
anti-permeability effect in the choroidal vascular bed. Given these
data, we believe that sonepcizumab has the potential to be a
monotherapy. The possibility of a synergistic effect with current
pan-VEGF-A blocking agents also exists.
Example 22
Reduction of Macrophage Infiltration in the Retina after Treatment
with Antibody to S1P
Age-related macular degeneration (AMD) is a disease associated with
aging that gradually destroys sharp, central vision. There are two
main types of macular degeneration. The dry or atrophic form which
accounts for 85-90% of AMD cases, and the wet form of AMD
characterized by the growth of abnormal blood vessels. Dry macular
degeneration is diagnosed when yellowish spots known as drusen
begin to accumulate from deposits or debris from deteriorating
tissue primarily in the area of the macula. Gradual central vision
loss may occur. There is no effective treatment for the most
prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by
abnormalities in the retinal pigment epithelium (RPE) that lies
beneath the photoreceptor cells and normally provides critical
metabolic support to these light-sensing cells. Secondary to RPE
dysfunction, macular rods and cones degenerate leading to the
irreversible loss of vision. Oxidative stress, ischemia, formation
of drusen, accumulation of lipofuscin, local inflammation and
reactive gliosis represent the pathologic processes implicated in
pathogenesis of atrophic AMD. Of these processes, inflammation is
emerging as a key contributor to tissue damage. Macrophage
infiltration into the macula of patients with dry AMD has been
demonstrated to be an important component of the damaging
inflammatory response. Therefore an agent which could mitigate
macrophage infiltration would be a valuable therapeutic, as
inhibition of macrophage infiltration would likely diminish macular
tissue damage. Such an agent may also decrease the rate at which
dry AMD converts to wet AMD.
In a model of ischemic and inflammatory retinopathy, a 55%
inhibition of macrophage infiltration has now been demonstrated
after treatment with an anti S1P antibody. These data were
generated using the well established murine oxygen induced
retinopathy model (also known as the retinopathy of prematurity
(ROP) model). Specifically, C57BL/6 mice were placed in 75% oxygen
on day 7 of life and at day 12 of life were returned to room air
and given an intraocular injection of 3 .mu.g of humanized anti S1P
antibody (LT1009, Sonepcizumab.TM.) in one eye and vehicle in the
fellow eye. At day 17 of life, the mice received an intraocular
injection of FITC-labeled antibody to F4/80 (a pan-macrophage
marker) and after 8 hours, mice were euthanized. The globes were
removed and fixed in PBS-buffered formalin at room temperature for
5 hours. Retinas were dissected and washed with phosphate-buffered
saline containing 0.25% Triton X-100 and whole mounted. Slides were
viewed with a Nikon Fluorescence Microscope and retinal macrophages
were quantified. The results are shown in Table 8 below.
TABLE-US-00010 TABLE 8 Reduction in macrophage infiltration in the
retina by treatment with humanized monoclonal antibody to S1P # of
macrophages % reduction in per retina macrophage density Saline
control S1P antibody Saline control S1P antibody 2513 .+-. 115 1136
.+-. 33 100 .+-. 0.5 55.4 .+-. 1.3 P < 0.001 P < 0.0001
On the basis of these data and the known role of macrophages in the
pathogenesis of dry AMD it is believed that anti-S1P antibodies
represent an effective therapeutic agent for the treatment of dry
AMD.
Example 23
Response of SC COLO205 Colorectal Tumor Xenograft in Nude NCr Mice
to Treatment with 25-75 mg/kg LT1009, Alone and in Combination with
Avastin or Paclitaxel
The objective of this study was to determine the efficacy of
LT1009, alone and in combination with other anti-cancer agents, to
retard the progression of human colorectal (COLO0205) carcinoma
tumors grafted subcutaneous (sc) and established in female Ncr
(nu/nu) mice.
Nude mice were implanted sc near the right flank with one fragment
per mouse of COLO 205 tumor from an in vivo passage. All treatments
were initiated the day when 60 mice in each experiment established
tumors ranging in size from approximately 100 to 200 mm3. The mice
(n=10 per group) were then treated with either 25 mg/kg of LT1009,
50 mg/kg LT1009, 40 mg/kg Avastin, 50 mg/kg LT1009 plus 40 mg/kg
Avastin, 15 mg/kg Paclitaxel or vehicle (saline). 25 or 50 mg/kg
LT1009 and saline were administered ip once q3d in a volume of 0.1
mL/20 g body weight for the duration of the experiment. Avastin was
administered iv at a dosage of 40 mg/kg/dose on a q7d schedule,
injected in a volume of 0.1 mL/20 g body weight. Paclitaxel
(positive control), was administered iv at a dosage of 15
mg/kg/dose on a q1d.times.5 schedule, injected in a volume of 0.1
mL/10 g body weight. On Day 21, the dose of 25 mg/kg LT1009 was
increased to 75 mg/kg LT1009 for the duration for the study.
Animals were observed daily for mortality. Tumor dimensions and
body weights were collected twice weekly starting with the first
day of treatment and including the day of study termination. When
the median tumor in the vehicle-treated control group in each study
reached approximately 4,000 mg, the study was terminated. Tumors
from each animal were harvested, wet weights were recorded, tumors
were processed for determination of microvascular densities (MVD)
by CD-31 staining. Tumor weights (mg) were calculated using the
equation for an ellipsoid sphere (l.times.w.sup.2)/2=mm.sup.3,
where l and w refer to the larger and smaller dimensions collected
at each measurement and assuming unit density (1 mm.sup.3=1
mg).
TABLE-US-00011 TABLE 9 Numerical summary of findings -Colo205 %
Reduction Final Tumor Compared to Vehicle- Treatment Weights (mg)
Treated Mice Vehicle 3047.25 -- 50 mg/kg LT1009 2071.17 32% 25/75
mg/kg 2465.60 20% LT1009 Avastin 1967.90 35% Avastin + 50 mg/kg
1614.40 48% LT1009 Paclitaxel 0 100%
50 mg/kg LT1009 substantially inhibited tumor progression
(p<0.018), as measured by final tumor weights, by 32% when
compared to tumors from saline-treated animals. 25/75 mg/kg LT1009
was also effective in reducing final tumor weights by 20%; however,
this reduction was not statistically significant. 50 mg/kg LT1009
was as effective as Avastin in reducing final tumor weights (32%
versus 35% reduction, respectively). The combination of LT1009 and
Avastin was more effective than either agent alone, demonstrating a
48% reduction in tumor weights when compared to saline-treated
animals. Thus the effects of LT1009 and Avastin appear to be
additive. The positive control, Paclitaxel, completely eliminated
the pre-established tumors.
Example 24
Response of SC HT29 Colorectal Tumor Xenograft in Nude NCr Mice to
Treatment with 50 mg/kg LT1009, Alone and in Combination with
Avastin and 5-FU
The objective of this study is to evaluate the antitumor efficacy
of LT1009, alone and in combination with other anti-cancer agents,
against human HT29 colon tumor xenografts implanted sc in female
athymic NCr-nu/nu mice.
Nude mice were implanted sc near the right flank with one fragment
per mouse of HT29 tumor from an in vivo passage. All treatments
were initiated the day when 60 mice in each experiment established
tumors ranging in size from approximately 100 to 200 mm.sup.3.
There were ten mice per treatment group. 50 mg/kg LT1009 and saline
were administered ip q2d in a volume of 0.1 mL/20 g body weight for
the duration of the experiment. 75 mg/kg 5-FU and 20 mg/kg Avastin
were administered ip and iv at a dosage of 75 mg/kg/dose and 20
mg/kg/dose, respectively, q4d, injected in a volume of 0.1 mL/10 g
body weight. The first dose of LT1009 consisted of 100 mg/kg
administered iv.
Animals were observed daily for mortality. Tumor dimensions and
body weights were collected twice weekly starting with the first
day of treatment and including the day of study termination. When
the median tumor in the vehicle-treated control group in each study
reached approximately 4,000 mg, the study was terminated. Tumors
from each animal were harvested, wet weights were recorded, and
tumors were processed for determination of MVD by CD-31 staining.
Tumor weights (mg) were calculated using the equation for an
ellipsoid sphere (l.times.w.sup.2)/2=mm.sup.3, where l and w refer
to the larger and smaller dimensions collected at each measurement
and assuming unit density (1 mm.sup.3=1 mg).
TABLE-US-00012 TABLE 10 Final Tumor Weights- HT29 Final Tumor %
Reduction Weights Significance compared to Vehicle- Treatment (mg)
(p-value) Treated Mice Vehicle 2723.67 -- -- LT1009 2390.63 1.00
13% Avastin 1927.44 0.39 30% LT1009 + Avastin 1624.90 0.001 41%
5-FU 1963.71 0.099 28% LT1009 + 5-FU 1948.00 0.049 29%
50 mg/kg LT1009 reduced tumor progression, as measured by tumor
weights, by 13% while Avastin reduced tumor weights by 30% when
compared to tumors from saline-treated animals. The combination of
LT1009 and Avastin was more effective than either agent alone
demonstrating a statistically significant 41% reduction in tumor
weights when compared to saline-treated animals. Treatment with
5-FU reduced tumor weights by 28%. 5-FU showed minimal additive
effect with LT1009 demonstrating a 29% inhibition of final tumor
weights.
Example 25
Response of SC DU145 Prostate Tumor Xenograft in Nude NCr Mice to
Treatment with 50 mg/kg LT1009, Alone or in Combination with
Avastin or Paclitaxel
The objective of this study was to determine the efficacy of
LT1009, alone and in combination with other anti-cancer agents, to
retard the progression of human prostate (DU145) carcinoma tumors
grafted subcutaneous (sc) and established in female Ncr (nu/nu)
mice.
Nude mice were implanted sc near the right flank with one fragment
per mouse of DU145 tumor from an in vivo passage. All treatments
were initiated the day when 60 mice in each experiment established
tumors ranging in size from approximately 100 to 200 mm.sup.3. The
mice (n=10/group) were then treated with either 50 mg/kg of LT1009,
20 mg/kg Avastin, 7.5 mg/kg Paclitaxel, 50 mg/kg LT1009 plus 20
mg/kg Avastin, 50 mg/kg LT1009 plus 7.5 mg/kg Paclitaxel or vehicle
(saline). 50 mg/kg LT1009 and saline were administered ip q2d in a
volume of 0.1 mL/20 g body weight for the duration of the
experiment. Paclitaxel and Avastin were administered iv and ip at a
dosage of 7.5 mg/kg/dose and 20 mg/kg/dose, q1d.times.5 and q4d,
respectively, injected in a volume of 0.1 mL/10 g body weight. The
first dose of LT1009 consisted of 100 mg/kg administered iv.
During the course of the study tumor growth was monitored by
measuring the sc tumors on three axes and calculating the volume.
At the end of the study final tumor weights and volumes were
determined and then the mice were sacrificed, the tumors harvested.
Microvascular densities (MVD) of the tumors were then determined by
CD-31 staining.
TABLE-US-00013 TABLE 11 Numerical summary of findings- DU145 Final
Tumor % Reduction Weights Significance compared to Vehicle-
Treatment (mg) (p-value) Treated Mice Vehicle 2703 -- -- LT1009
2242 0.00 28% Avastin 578 0.00 79% LT1009 + Avastin 676 0.00 75%
Paclitaxel 539 0.00 80% LT1009 + Paclitaxel 373 0.00 84%
50 mg/kg LT1009 significantly (p<0.00) reduced tumor
progression, as measured by final tumor weights, by 28%. Avastin
and Paclitaxel also significantly (p<0.00) reduced final tumor
weights by 80% when compared to tumors from saline-treated animals.
LT1009 did not significantly increase the anti-tumor activity, as
measured by final tumor volumes, of Avastin or Paclitaxel.
Example 26
Response of RPMI 8226 Human Myeloma Tumor Xenograft in CB17 SCID
Mice to Treatment with 25 ml/kg or 50 mg/kg LT1009, Alone and in
Combination with Bortezomib
The objective of this study is to evaluate the antitumor efficacy
of LT1009, alone and in combination with the anti-cancer agent
Bortezomib, against human RPMI human myeloma tumor xenografts
implanted sc in female CB17 SCID mice.
Nude mice (CB17 SCID, aged 4-5 weeks, weight 18-22 gm, female mice
obtained from Harlan) were injected sc with RPMI 8226 cells
harvested from tissue culture (.about.1.times.10.sup.7
cells/mouse). When tumors grew to approximately 100 mm3 in size,
animals were pair-matched by tumor size into treatment and control
groups (10 mice per group). Initial dosing began Day 1 following
pair-matching. Animals in all groups were dosed by weight (0.01 ml
per gram; 10 ml/kg). LT1009 in vehicle was administered by
intraperitoneal (IP) injection once every three days until study
completion (Q3D to end). Bortezomib was administered by intravenous
injection via tail vein once every three days for six treatments
(Q3D.times.6). To serve as a negative control, LT1009 vehicle (0.9%
saline) was administered IP on a Q3D to end schedule.
Individual and group mean tumor volumes .+-.SEM are recorded twice
weekly until study completion beginning Day 1. Final mean tumor
volume .+-.SEM for each group are reported at study completion;
animals experiencing partial or complete tumor regressions or
animals experiencing technical or drug-related deaths are censored
from these calculations.
TABLE-US-00014 TABLE 12 Final Tumor Volumes- RPMI % Reduction Final
Tumor compared to Vehicle- Treatment Weights (mg) Treated Mice
Vehicle 2083 0 Bortezomib 1664 20% 25 mg/kg LT1009 1860 11% 50
mg/kg LT1009 1978 5% 50 mg/kg LT1009 + 1832 12% Bortezomib
All of the compositions and methods described and claimed herein
can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the compositions and methods. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit and scope of the invention as
defined by the appended claims.
All patents, patent applications, and publications mentioned in the
specification are indicative of the levels of those of ordinary
skill in the art to which the invention pertains. All patents,
patent applications, and publications, including those to which
priority or another benefit is claimed, are herein incorporated by
reference to the same extent as if each individual publication was
specifically and individually indicated to be incorporated by
reference.
The invention illustratively described herein suitably may be
practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of", and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
SEQUENCE LISTINGS
1
53126DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1atggratgga gckggrtctt tmtctt 26221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2cagtggatag acagatgggg g 21321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3cagtggatag accgatgggg c
21421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4cagtggatag actgatgggg g 21521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5caagggatag acagatgggg c 21618DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gtctctgatt ctagggca
18720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7actggatggt gggaagatgg 208120PRTMus sp. 8Gln Ala
His Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser
Val Lys Ile Ser Cys Lys Val Ser Gly Phe Ile Phe Ile Asp His 20 25
30Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45Gly Cys Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn Glu Met
Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr
Ala Tyr65 70 75 80Ile Gln Val Asn Ser Leu Thr Phe Glu Asp Ser Ala
Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp
Phe Asp Phe Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser 115
1209107PRTMus sp. 9Glu Thr Thr Val Thr Gln Ser Pro Ala Ser Leu Ser
Met Ala Ile Gly1 5 10 15Glu Lys Val Thr Ile Arg Cys Ile Thr Thr Thr
Asp Ile Asp Asp Asp 20 25 30Met Asn Trp Phe Gln Gln Lys Pro Gly Glu
Pro Pro Asn Leu Leu Ile 35 40 45Ser Glu Gly Asn Ile Leu Arg Pro Gly
Val Pro Ser Arg Phe Ser Ser 50 55 60Ser Gly Tyr Gly Thr Asp Phe Leu
Phe Thr Ile Glu Asn Met Leu Ser65 70 75 80Glu Asp Val Ala Asp Tyr
Tyr Cys Leu Gln Ser Asp Asn Leu Pro Phe 85 90 95Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys 100 1051011PRTMus sp. 10Ile Thr Thr Thr Asp
Ile Asp Asp Asp Met Asn1 5 10117PRTMus sp. 11Glu Gly Asn Ile Leu
Arg Pro1 5129PRTMus sp. 12Leu Gln Ser Asp Asn Leu Pro Phe Thr1
5135PRTMus sp. 13Asp His Thr Ile His1 51417PRTMus sp. 14Cys Ile Ser
Pro Arg His Asp Ile Thr Lys Tyr Asn Glu Met Phe Arg1 5 10
15Gly1512PRTMus sp. 15Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp
Phe1 5 1016147PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 16Met Gly Ser Thr Ala Ile Leu Ala
Leu Leu Leu Ala Val Leu Gln Gly1 5 10 15Val Cys Ser Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser Leu Lys
Ile Ser Cys Gln Ser Phe Gly Tyr Ile Phe 35 40 45Ile Asp His Thr Ile
His Trp Val Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp Met Gly
Cys Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75 80Glu Met
Phe Arg Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser 85 90 95Thr
Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105
110Tyr Phe Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp
115 120 125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
Thr Lys 130 135 140Gly Pro Ser14517134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Leu Thr Gln Ser Pro Ser
Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile
Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn Trp Tyr Gln Gln Glu
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Gly Asn Ile Leu
Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125Lys Arg Glu Trp Ile
Pro 1301833DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 18ataaccacca ctgatattga tgatgatatg aac
331921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19gaaggcaata ttcttcgtcc t
212027DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 20ttgcagagtg ataacttacc attcacg
272113DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 21gaccatactt cac 132251DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22tgtatttctc ccagacatga tattactaaa tacaatgaga
tgttcagggg c 512336DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 23ggggggttct acggtagtac
tatctggttt gacttt 362451DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 24gctatttctc
ccagacatga tattactaaa tacaatgaga tgttcagggg c 5125455DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
25cgccaagctt gccgccacca tggggtcaac cgccatcctc gccctcctcc tggctgttct
60ccaaggagtc tgttccgagg tgcagctggt gcagtctgga gcagaggtga aaaagcccgg
120ggagtctctg aagatctcct gtcagagttt tggatacatc tttatcgacc
atacttcact 180gggtgcgcca gatgcccggg caaggcctgg agtggatgtg
tatttctccc agacatgata 240ttactaaata caatgagatg ttcaggggcc
aggtcaccat ctcagccgac aagtccagca 300gcaccgccta cttgcagtgg
agcagcctga aggcctcgga caccgccatg tatttctgtg 360cgagaggggg
gttctacggt agtactatct ggtttgactt ttggggccaa gggacaatgg
420tcaccgtctc ttcagcctcc accaagggcc catcg 45526452DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
26cgccaagctt gccgccacca tggggtcaac cgccatcctc gccctcctcc tggctgttct
60ccaaggagtc tgttccgagg tgcagctggt gcagtctgga gcagaggtga aaaagcccgg
120ggagtctctg aagatctcct gtcagagttt tggatacatc gaccatactt
cactggatgc 180gccagatgcc cgggcaaggc ctggagtgga tgggggctat
ttctcccaga catgatatta 240ctaaatacaa tgagatgttc aggggccagg
tcaccatctc agccgacaag tccagcagca 300ccgcctactt gcagtggagc
agcctgaagg cctcggacac cgccatgtat ttctgtgcga 360gaggggggtt
ctacggtagt actatctggt ttgacttttg gggccaaggg acaatggtca
420ccgtctcttc agcctccacc aagggcccat cg 45227147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1
5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr
Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met Arg Gln Met Pro Gly
Gln Gly Leu 50 55 60Glu Trp Met Gly Ala Ile Ser Pro Arg His Asp Ile
Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly
Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro
Ser14528419DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 28cgccaagctt gccgccacca tggacatgag
ggtccccgct cagctcctgg ggctcctgct 60gctctggctc ccaggtgcca gatgtgaaac
gacactcacg cagtctccat ccttcctgtc 120tgcatctgta ggagacagag
tcaccatcac ataaccacca ctgatattga tgatgatatg 180aactggtatc
agcaggaacc agggaaagcc cctaagctcc tgatctatga aggcaatatt
240cttcgtcctg gggtcccatc aaggttcagc ggcagtggat ctggcacaga
tttcactctc 300accatcagca aattgcagcc tgaagatttt gcaacttatt
actgtttgca gagtgataac 360ttaccattca cgttcggcca agggaccaag
ctggagatca aacgtgagtg gatcccgcg 41929407DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
29cgccaagctt gccgccacca tggacatgag ggtccccgct cagctcctgg ggctcctgct
60gctctggctc ccaggggcca gatgtgaaac gacagtgacg cagtctccat ccttcctgtc
120tgcatctgta ggagacagag tcaccatcac ttgcataacc accactgata
ttgatgatga 180tatgaactgg ttccagcagg aaccagggaa agcccctaag
ctcctgatct ccgaaggcaa 240tattcttcgt cctggggtcc catcaagatt
cagcagcagt ggatatggca cagatttcac 300tctcaccatc agcaaattgc
agcctgaaga ttttgcaact tattactgtt tgcagagtga 360taacttacca
ttcactttcg gccaagggac caagctggag atcaaac 40730126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1
5 10 15Gly Ala Arg Cys Glu Thr Thr Val Thr Gln Ser Pro Ser Phe Leu
Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile Thr Thr
Thr Asp 35 40 45Ile Asp Asp Asp Met Asn Trp Phe Gln Glu Pro Gly Lys
Ala Pro Lys 50 55 60Leu Leu Ile Ser Glu Gly Asn Ile Leu Arg Pro Gly
Val Pro Ser Arg65 70 75 80Phe Ser Ser Ser Gly Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Lys 85 90 95Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln Ser Asp Asn 100 105 110Leu Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 115 120 1253117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Ala
Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn Glu Met Phe Arg1 5 10
15Gly32140PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu
Ser Val Thr Thr Gly1 5 10 15Val His Ser Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys
Gln Ser Phe Gly Tyr Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met
Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp Met Gly Ala Ile Ser
Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe
Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120
125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135
14033127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg Cys Glu Thr Thr Val Thr Gln
Ser Pro Ser Phe Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Ile Thr Thr Thr Asp 35 40 45Ile Asp Asp Asp Met Asn Trp Phe
Gln Gln Glu Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Ser Glu Gly
Asn Ile Leu Arg Pro Gly Val Pro Ser65 70 75 80Arg Phe Ser Ser Ser
Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Lys Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ser Asp 100 105 110Asn Leu
Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120
125342034DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 34aagcttgccg ccaccatgga atggagctgg
gtgttcctgt tctttctgtc cgtgaccaca 60ggcgtgcatt ctgaggtgca gctggtgcag
tctggagcag aggtgaaaaa gcccggggag 120tctctgaaga tctcctgtca
gagttttgga tacatcttta tcgaccatac tattcactgg 180atgcgccaga
tgcccgggca aggcctggag tggatggggg ctatttctcc cagacatgat
240attactaaat acaatgagat gttcaggggc caggtcacca tctcagccga
caagtccagc 300agcaccgcct acttgcagtg gagcagcctg aaggcctcgg
acaccgccat gtatttctgt 360gcgagagggg ggttctacgg tagtactatc
tggtttgact tttggggcca agggacaatg 420gtcaccgtct cttcagcctc
caccaagggc ccatcggtct tccccctggc accctcctcc 480aagagcacct
ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa
540ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac
cttcccggct 600gtcctacagt cctcaggact ctactccctc agcagcgtgg
tgaccgtgcc ctccagcagc 660ttgggcaccc agacctacat ctgcaacgtg
aatcacaagc ccagcaacac caaggtggac 720aagagagttg gtgagaggcc
agcacaggga gggagggtgt ctgctggaag ccaggctcag 780cgctcctgcc
tggacgcatc ccggctatgc agtcccagtc cagggcagca aggcaggccc
840cgtctgcctc ttcacccgga ggcctctgcc cgccccactc atgctcaggg
agagggtctt 900ctggcttttt ccccaggctc tgggcaggca caggctaggt
gcccctaacc caggccctgc 960acacaaaggg gcaggtgctg ggctcagacc
tgccaagagc catatccggg aggaccctgc 1020ccctgaccta agcccacccc
aaaggccaaa ctctccactc cctcagctcg gacaccttct 1080ctcctcccag
attccagtaa ctcccaatct tctctctgca gagcccaaat cttgtgacaa
1140aactcacaca tgcccaccgt gcccaggtaa gccagcccag gcctcgccct
ccagctcaag 1200gcgggacagg tgccctagag tagcctgcat ccagggacag
gccccagccg ggtgctgaca 1260cgtccacctc catctcttcc tcagcacctg
aactcctggg gggaccgtca gtcttcctct 1320tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc acatgcgtgg 1380tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg
1440aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg
taccgtgtgg 1500tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac aagtgcaagg 1560tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc aaaggtggga 1620cccgtggggt gcgagggcca
catggacaga ggccggctcg gcccaccctc tgccctgaga 1680gtgaccgctg
taccaacctc tgtccctaca gggcagcccc gagaaccaca ggtgtacacc
1740ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg
cctggtcaaa 1800ggcttctatc ccagcgacat cgccgtggag tgggagagca
atgggcagcc ggagaacaac 1860tacaagacca cgcctcccgt gctggactcc
gacggctcct tcttcctcta tagcaagctc 1920accgtggaca agagcaggtg
gcagcagggg aacgtcttct catgctccgt gatgcatgag 1980gctctgcaca
accactacac gcagaagagc ctctccctgt ctccgggtaa atag
203435455PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 35Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu
Ser Val Thr Thr Gly1 5 10 15Val His Ser Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys
Gln Ser Phe Gly Tyr Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met
Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp Met Gly Ala Ile Ser
Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe
Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120
125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly145 150 155 160Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210
215 220Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Ala
Pro225 230 235 240Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 260 265 270Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp305 310 315 320Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330
335Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys 355 360 365Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 370 375 380Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys385 390 395 400Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445Leu
Ser Leu Ser Pro Gly Lys 450 45536720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
36aagcttgccg ccaccatgtc tgtgcctacc caggtgctgg gactgctgct gctgtggctg
60acagacgccc gctgtgaaac gacagtgacg cagtctccat ccttcctgtc tgcatctgta
120ggagacagag tcaccatcac ttgcataacc accactgata ttgatgatga
tatgaactgg 180ttccagcagg aaccagggaa agcccctaag ctcctgatct
ccgaaggcaa tattcttcgt 240cctggggtcc catcaagatt cagcagcagt
ggatatggca cagatttcac tctcaccatc 300agcaaattgc agcctgaaga
ttttgcaact tattactgtt tgcagagtga taacttacca 360ttcactttcg
gccaagggac caagctggag atcaaacgta cggtggctgc accatctgtc
420ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 480ctgaataact tctatcccag agaggccaaa gtacagtgga
aggtggataa cgccctccaa 540tcgggtaact cccaggagag tgtcacagag
caggacagca aggacagcac ctacagcctc 600agcagcaccc tgacgctgag
caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660gtcacccatc
agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag
72037234PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg Cys Glu Thr Thr Val Thr Gln
Ser Pro Ser Phe Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Ile Thr Thr Thr Asp 35 40 45Ile Asp Asp Asp Met Asn Trp Phe
Gln Gln Glu Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Ser Glu Gly
Asn Ile Leu Arg Pro Gly Val Pro Ser65 70 75 80Arg Phe Ser Ser Ser
Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Lys Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ser Asp 100 105 110Asn Leu
Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 115 120
125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys225 23038451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
38Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1
5 10 15Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr Ile Phe Ile Asp
His 20 25 30Thr Ile His Trp Met Arg Gln Met Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn
Glu Met Phe 50 55 60Arg Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Phe Cys 85 90 95Ala Arg Gly Gly Phe Tyr Gly Ser Thr
Ile Trp Phe Asp Phe Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 45039147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1
5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr
Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met Arg Gln Met Pro Gly
Gln Gly Leu 50 55 60Glu Trp Met Gly Cys Ile Ser Pro Arg His Asp Ile
Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly
Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro
Ser14540147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 40Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu
Ala Val Leu Gln Gly1 5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys
Gln Ser Phe Gly Phe Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met
Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Cys Ile Ser
Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe
Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120
125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140Gly Pro Ser14541147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Met Gly Ser Thr Ala Ile
Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1 5 10 15Val Cys Ser Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser
Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr Ile Phe 35 40 45Ile Asp His
Thr Ile His Trp Val Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp
Ile Gly Cys Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75
80Glu Met Phe Arg Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser
85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile
Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr Lys 130 135 140Gly Pro Ser14542147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1
5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Phe
Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Val Arg Gln Met Pro Gly
Gln Gly Leu 50 55 60Glu Trp Met Gly Cys Ile Ser Pro Arg His Asp Ile
Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly
Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro
Ser14543147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 43Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu
Ala Val Leu Gln Gly1 5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys
Gln Ser Phe Gly Phe Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met
Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Cys Ile Ser
Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly
Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe
Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120
125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140Gly Pro Ser14544147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Met Gly Ser Thr Ala Ile
Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1 5 10 15Val Cys Ser Glu Ala
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Glu Ser
Leu Lys Ile Ser Cys Gln Ser Phe Gly Phe Ile Phe 35 40 45Ile Asp His
Thr Ile His Trp Met Arg Gln Met Pro Gly Gln Gly Leu 50 55 60Glu Trp
Ile Gly Cys Ile Ser Pro Arg His Asp Ile Thr Lys Tyr Asn65 70 75
80Glu Met Phe Arg Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly Phe Tyr Gly Ser Thr Ile
Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr Lys 130 135 140Gly Pro Ser14545147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1
5 10 15Val Cys Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr
Ile Phe 35 40 45Ile Asp His Thr Ile His Trp Met Arg Gln Met Pro Gly
Gln Gly Leu 50 55 60Glu Trp Met Gly Ala Ile Ser Pro Arg His Asp Ile
Thr Lys Tyr Asn65 70 75 80Glu Met Phe Arg Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met 100 105 110Tyr Phe Cys Ala Arg Gly Gly
Phe Tyr Gly Ser Thr Ile Trp Phe Asp 115 120 125Phe Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro
Ser14546134PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Leu
Thr Gln Ser Pro Ser Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Ile Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn
Trp Tyr Gln Gln Glu Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Ser
Glu Gly Asn Ile Leu Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp
Asn Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120
125Lys Arg Glu Trp Ile Pro 13047134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Leu Thr Gln Ser Pro Ser
Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile
Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn Trp Phe Gln Gln Glu
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Gly Asn Ile Leu
Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125Lys Arg Glu Trp Ile
Pro 13048133PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 48Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg
Cys Glu Thr Thr Val Thr Gln Ser Pro Ser Phe 20 25 30Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Ile Thr Thr 35 40 45Thr Asp Ile
Asp Asp Asp Met Asn Trp Tyr Gln Gln Glu Pro Gly Lys 50 55 60Ala Pro
Lys Leu Leu Ile Ser Glu Gly Asn Ile Leu Arg Pro Gly Val65 70 75
80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile 115 120 125Lys Arg Glu Trp Ile 13049134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Leu Thr Gln Ser Pro Ser
Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile
Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn Trp Phe Gln Gln Glu
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Ser Glu Gly Asn Ile Leu
Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125Lys Arg Glu Trp Ile
Pro 13050132PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 50Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Glu
Thr Thr Val Thr Gln Ser Pro Ser Phe 20 25 30Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Ile Thr Thr 35 40 45Thr Asp Ile Asp Asp
Asp Met Asn Trp Phe Gln Gln Glu Pro Gly Lys 50 55 60Ala Pro Lys Leu
Leu Ile Ser Glu Gly Asn Ile Leu Arg Pro Gly Val65 70 75 80Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile
Ser Lys Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105
110Ser Asp Asn Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
115 120 125Lys Arg Glu Trp 13051132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Val Thr Gln Ser Pro Ser
Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile
Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn Trp Phe Gln Gln Glu
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Ser Glu Gly Asn Ile Leu
Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125Lys Arg Glu Trp
13052134PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 52Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Val
Thr Gln Ser Pro Ser Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Ile Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn
Trp Phe Gln Gln Glu Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Ser
Glu Gly Asn Ile Leu Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser
Ser Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp
Asn Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120
125Lys Arg Glu Trp Ile Pro 13053134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Glu Thr Thr Leu Thr Gln Ser Pro Ser
Phe 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ile
Thr Thr 35 40 45Thr Asp Ile Asp Asp Asp Met Asn Trp Tyr Gln Gln Glu
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Ser Glu Gly Asn Ile Leu
Arg Pro Gly Val65 70 75 80Pro Ser Arg Phe Ser Ser Ser Gly Tyr Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Lys Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln 100 105 110Ser Asp Asn Leu Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125Lys Arg Glu Trp Ile
Pro 130
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